Nitro-Oxidative Stress Correlates with Se Tolerance of Astragalus Species

Nitro-Oxidative Stress Correlates with Se Tolerance of Astragalus Species Abstract At high concentrations, selenium (Se) exerts phytotoxic effects in non-tolerant plant species partly due to the induction of nitro-oxidative stress; however, these processes are not fully understood. In order to obtain a more accurate view of the involvement of nitro-oxidative processes in plant Se sensitivity, this study aims to characterize and compare Se-triggered changes in reactive oxygen (ROS) and nitrogen species (RNS) metabolism and the consequent protein tyrosine nitration as a marker of nitrosative stress in the non-accumulator Astragalus membranaceus and the Se hyperaccumulator Astragalus bisulcatus. The observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth and cell viability) supported that A. membranaceus is Se sensitive while the hyperaccumulator A. bisulcatus tolerates high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism, leading to its accumulation. Furthermore, Se increased the production or disturbed the metabolism of RNS (nitric oxide, peroxynitrite and S-nitrosoglutathione), consequently resulting in intensified protein tyrosine nitration in sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se-induced ROS/RNS accumulation and tyrosine nitration proved to be negligible, suggesting that this species is able to prevent Se-induced nitro-oxidative stress. Introduction Selenium (Se) is a non-metal element which seems to be non-essential for higher plants. Still, its chemical similarity to sulfur (S) results in its uptake and metabolism via S transporters and pathways (Pilon-Smits and Quinn 2010). Moreover, a few plant species not only take up but also accumulate or hyperaccumulate high Se levels in their tissues. The ability for Se hyperaccumulation has been described in 45 plant taxa in six families (White 2016). The Astragalus (Fabaceae) genus is the most representative since a large number of species (25) in the genus have the ability to take up and tolerate high concentrations of Se (Shrift 1969). Species such as Astragalus bisulcatus grow on seleniferous soils and can accumulate >1,000 μg g−1 DW Se (up to 1% of its dry weight). Hyperaccumulators possess 10- to 100-fold higher endogenous Se content as well as a higher Se:S ratio compared with non-accumulators (White et al. 2007). Another distinctive feature of hyperaccumulators is the active sulfate/selenate assimilation which is suggested by the dominance of organic Se forms (γ-glutamyl-methyl-selenocysteine) in their tissues. Hyperaccumulators can be characterized by notable root to shoot Se translocation (Mehdawi and Pilon-Smits 2012). Species such as A. bisulcatus are able to sequester Se in their epidermis and trichomes, which may have a role both in defense and Se stress mitigation (Freeman et al. 2006). The mechanism responsible for Se hyperaccumulation is the constitutive expression of several SULTR transporters, which contributes to the preferential uptake of selenate over sulfate (Cabannes et al. 2011). Also, the expression of certain enzymes involved in selenate/sulfate assimilation is enhanced in hyperaccumulators, resulting in greater inorganic–organic conversion (Freeman et al. 2010). Moreover, hyperaccumulators express selenocysteine methyltransferase (SMT) which is responsible for the conversion of toxic selenocysteine to methyl-selenocysteine (Sors et al. 2009). Se tolerance is also typical for hyperaccumulators; however, the molecular mechanism of this ability is only partly understood. High tissue concentrations of inorganic Se forms can induce the production of reactive oxygen species (ROS) such as superoxide (O2·–), hydrogen peroxide (H2O2) and hydroxyl radical (OH·), leading to oxidative stress (Van Hoewyk 2013). The amount of the generated ROS and consequently the redox homeostasis is precisely controlled by antioxidant mechanisms. Beyond the enzymatic components such as superoxide dismutase (SOD), catalase (CAT) and peroxidases (PODs), non-enzymatic antioxidants such as ascorbate and glutathione (GSH) play crucial role in the defense against oxidative damage (Das and Roychoudhury 2014). For Se-induced ROS accumulation, GSH and its depletion seems to be responsible (Van Hoewyk 2013). According to previous data, hyperaccumulators prefer to produce organic Se forms presumably in order to avoid oxidative stress (Freeman et al. 2006, Van Hoewyk 2013). Besides ROS, reactive nitrogen species (RNS) are also formed as the effect of environmental stresses such as Se exposure (reviewed by Kolbert et al. 2016). This group of nitric oxide (NO)-related molecules consists of peroxynitrite (ONOO–), S-nitrosoglutathione (GSNO), dinitrogen trioxide (N2O3), dinitrogen tetroxide (N2O4) and nitrogen dioxide radical (·NO2) (Corpas et al. 2007). The overproduction of RNS leads to nitrosative stress during which one of the principle mechanisms is the nitration of tyrosine residues in certain proteins yielding 3-nitrotyrosine (Corpas et al. 2013). This modification causes structural and functional changes in the affected proteins. In most published cases, tyrosine nitration results in loss of activity of the target plant proteins (Kolbert et al. 2017) or it can negatively affect signal transduction through the prevention of tyrosine phosphorylation (Galetskiy et al. 2011). An Se-induced increase in protein tyrosine nitration and in oxidative parameters (ROS levels, lipid peroxidation and antioxidants) has been revealed in the leaves of non-accumulator pea (Lehotai et al. 2016). Also, the relationship between the toxicity of Se forms and protein tyrosine nitration has been evaluated in the non-accumulator Arabidopsis thaliana and the secondary accumulator Brassica juncea (Molnár et al. 2018a, Molnár et al. 2018b), but there is no knowledge about RNS metabolism and protein nitration in Se hyperaccumulator plants such as A. bisulcatus. Another species in the Astragalus genus is Astragalus membranaceus, which is considered to be pharmacologically relevant. The root of this Astragalus species has been used in Chinese medicine for thousands of years because of its general strengthening effect. Based on the literature, in modern medicine it can provide perspectives for the prevention and therapy of cerebrovascular, cardiovascular, neurodegenerative and liver diseases (Yang et al. 2013). Despite the significance of A. membranaceus, we know little about its Se accumulation and tolerance or about reactive species metabolism and nitrosative stress. Therefore, this comparative study aims to explore the possible differences in Se-modified ROS and RNS metabolism and the consequent protein tyrosine nitration using the hyperaccumulator A. bisulcatus and A. membranaceus as another species in the same genus. The better understanding of tolerance mechanisms of Se hyperaccumulator plant species is of particular significance in phytoremediation (Gupta and Gupta 2017) and in biofortification (Wu et al. 2015) as well from an ecological (Schiavon and Pilon-Smits 2017) point of view. Furthermore, examination of Se accumulation and tolerance of the medicinal herb A. membranaceus can have importance in aspects of human health. Results Selenium uptake, accumulation and microelement imbalance Selenate-induced Se accumulation showed differences in the organs of Astragalus species (Fig. 1). In the root tissues of A. membranaceus, the Se concentration was significantly enhanced as an effect of increasing exogenous selenate supplementation (Fig. 1A). In the case of A. membranaceus cotyledons, Se accumulation was not concentration dependent and proved to be lower compared with the root (Fig. 1B). The Se content measured in cotyledons of 50 or 100 µM selenate-treated A. membranaceus did not reach the endogenous Se content of the control A. bisulcatus. The root of the hyperaccumulator A. bisulcatus showed moderate Se accumulation (Fig. 1A), while in the cotyledons a remarkable, concentration-dependent increase of Se content was observed (Fig. 1B). In the case of 100 µM selenate supplementation, the accumulated Se exceeded 1,700 µg g–1 DW in the cotyledons of A. bisulcatus. It has to be mentioned that a significant difference was observed in the endogenous Se contents of untreated Astragalus plants. Cotyledons of A. bisulcatus contained 200-fold more Se than the same organs of A. membranaceus (Fig. 1B). Regarding the root, a similar but much smaller (16-fold) difference was revealed (Fig. 1A). Fig. 1 View largeDownload slide Concentration of selenium in the root system (A) and in the cotyledons (B) of 14-day-old A. membranaceus and A. bisulcatus treated with 0 (control), 50 or 100 µM sodium selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Fig. 1 View largeDownload slide Concentration of selenium in the root system (A) and in the cotyledons (B) of 14-day-old A. membranaceus and A. bisulcatus treated with 0 (control), 50 or 100 µM sodium selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Selenate exposure led to the modification of microelement concentrations in the organs of Astragalus species (Table 1). Of the examined microelements, the contents of the essential Fe, Zn, Mn and B showed a notable reduction, especially in the cotyledons of A. membranaceus. However, the concentration of the above-mentioned elements was not affected at all or only slightly changed by selenate in A. bisulcatus cotyledons. Regarding the root system, more serious effects were observed in the case of A. membranaceus compared with A. bisulcatus, e.g. the Fe concentration decreased by 30% in A. membranaceus but only by 15% in A. bisulcatus. In contrast to the other microelements, Mo concentrations in A. membranaceus organs significantly increased as an effect of Se treatments. In A. bisulcatus, the concentrations of Mo were decreased or were not modified by Se (Table 1). Table 1 Concentrations of Fe, Zn, Mn, B and Mo in the root system and cotyledons of 14-day-old Astragalus species treated with 0, 50 or 100 µM selenate for 14 d Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Different superscript letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Table 1 Concentrations of Fe, Zn, Mn, B and Mo in the root system and cotyledons of 14-day-old Astragalus species treated with 0, 50 or 100 µM selenate for 14 d Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Different superscript letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Growth and Se tolerance of Astragalus species Se tolerance of Astragalus species was evaluated by germination capacity, biomass production, root meristem viability and root elongation on selenate-supplemented medium. Both species showed approximately 85% germination under control conditions, and this good germination capability was retained by A. bisulcatus on 50 and 100 µM selenate-treated plates (Fig. 2A). In contrast, the presence of selenate significantly and concentration dependently reduced the germination percentage of A. membranaceus. In the case of 100 µM Se treatment, 55% of A. membranaceus seeds placed on the medium were germinated, while A. bisulcatus showed better (∼70%) germination performance. Fig. 2 View largeDownload slide (A) Germination percentage of Astragalus species on agar media supplemented with 0 (control), 50 or 100 µM sodium selenate. Shoot (B) and root (C) fresh weight of 14-day-old A. membranaceus and A. bisulcatus plants treated with 0 (control), 50 or 100 µM selenate. Different letters indicate significant differences according to Duncan’s test (n = 15, P ≤ 0.05). (D) Representative images showing 14-day-old A. membranaceus and A. bisulcatus plants grown on control or 50 or 100 µM selenate-containing agar media. Photographs show three representative individuals per treatment. Scale bars = 3 cm. Fig. 2 View largeDownload slide (A) Germination percentage of Astragalus species on agar media supplemented with 0 (control), 50 or 100 µM sodium selenate. Shoot (B) and root (C) fresh weight of 14-day-old A. membranaceus and A. bisulcatus plants treated with 0 (control), 50 or 100 µM selenate. Different letters indicate significant differences according to Duncan’s test (n = 15, P ≤ 0.05). (D) Representative images showing 14-day-old A. membranaceus and A. bisulcatus plants grown on control or 50 or 100 µM selenate-containing agar media. Photographs show three representative individuals per treatment. Scale bars = 3 cm. With regard to biomass production, 14-day-old, untreated individuals of the species possessed a similar shoot weight (Fig. 2B). However, the root fresh weight of control A. membranaceus was significantly smaller (Fig. 2C) and the phenotype of the root system notably differed from that of A. bisulcatus (Fig. 2D). Both concentrations of exogenous Se (50 and 100 µM) negatively affected shoot (40% and 46% reduction, respectively) and root growth (57% and 75% reduction, respectively) of A. membranaceus (Fig. 2B, C) and a brown discoloration was visible on the root surface of Se-treated plants (Fig. 2D). In contrast, A. bisulcatus showed significantly slighter growth inhibition, since the root biomass was affected only by the highest Se dose (30% reduction) and none of the treatments inhibited shoot growth (Fig. 2B–D). Se tolerance correlates with the capability of maintaining primary root elongation; therefore, the Se tolerance index can be calculated from primary root length data (Tamaoki et al. 2008). Compared with the 100% tolerance of the untreated plants (indicated by a dashed line in Fig. 3A), 50 or 100 µM selenate resulted in a 35% or 25% tolerance index of A. membranaceus, respectively (Fig. 3A). However, A. bisulcatus was able to maintain its root growth, and Se even slightly increased elongation, resulting in tolerance indexes around or above 100%. Furthermore, we examined the Se tolerance of the species by evaluating the viability of the root meristem cells using fluorescein diacetate (FDA) staining (Fig. 3B, C). As expected from the previous data, the meristem cells of A. membranaceus showed 50% or 85% viability loss as the effect of 50 or 100 µM selenate exposure, respectively. Even though root elongation of A. bisulcatus was not negatively affected by any of the applied Se doses (Fig. 3A), root meristem cells suffered 50% viability loss as the effect of the highest Se concentration (Fig. 3B, C). We acknowledge that the use of plant tissues with highly reduced viability might limit the reliability of the data. At the same time, the choice of the 14 d treatment period proved to be necessary for the appearance of the effect, as well as for the emergence of tolerance in this comparative Astragalus system (Supplementary Fig. S1). Fig. 3 View largeDownload slide (A) Selenium tolerance indexes (%) of Astragalus species treated with 50 or 100 µM selenate for 14 d. The 100% tolerance index of untreated plants is indicated by a dashed line. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Viability of primary root meristem cells in control and selenate-treated Astragalus species. Significant differences were determined by Student’s t-test and indicated by asterisks (n = 15, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.= non-significant). (C) Representative microscopic images indicating root tips of control (C) and selenate-treated Astragalus species stained with fluorescein diacetate. Scale bars = 500 µm. Fig. 3 View largeDownload slide (A) Selenium tolerance indexes (%) of Astragalus species treated with 50 or 100 µM selenate for 14 d. The 100% tolerance index of untreated plants is indicated by a dashed line. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Viability of primary root meristem cells in control and selenate-treated Astragalus species. Significant differences were determined by Student’s t-test and indicated by asterisks (n = 15, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.= non-significant). (C) Representative microscopic images indicating root tips of control (C) and selenate-treated Astragalus species stained with fluorescein diacetate. Scale bars = 500 µm. Se-induced tissue-level changes in the roots To evaluate the Se-induced tissue-level changes in the root structure of both Astragalus species, we measured the diameter of the root, the thickness of the cortex and the diameter of the vascular cylinder (stele). Both untreated and Se-treated A. membranaceus plants had thick roots, and Se application did not significantly affect root diameter (F = 1.25, P = 0.29). In the case of control and 50 µM Se-treated plants, A. membranaceus had nearly twice as thick roots as A. bisulcatus (Fig. 4A). When 100 µM Se was added to the media, the roots of A. bisulcatus exhibited remarkable thickening, the degree of was similar to that of A. membranaceus. This tendency was also confirmed by analysis of correlation (r = 0.82, P < 0.001). Similarly, the sensitive A. membranaceus had a significantly thicker root cortex than the Se hyperaccumulator A. bisulcatus in both control and 50 µM Se-treated plants, but it was almost the same in the roots of 100 µM Se-treated plants of both species (Fig. 4B). Increasing Se levels significantly enhanced the thickness of the cortex in the case of A. bisulcatus (F = 403.88, P < 0.001; r = 0.88, P < 0.001), while a remarkable increase was found only in the root cortex of 50 µM Se-treated A. membranaceus (F = 33.88; P < 0.001; r = 0.34, P < 0.001). There was a remarkable increase of stele diameter in A. membranaceus roots exposed to 50 µM Se, while it significantly decreased compared with control after 100 µM Se application. The size of the stele in the roots of A. bisulcatus was only affected by the highest Se stress (Fig. 4C). The stele of control and 50 µM Se-treated A. membranaceus roots was at least twice as thick as that of A. bisulcatus. Se stress-induced deposition of callose was investigated in aniline blue-stained root sections taken from the mature zone. Significantly higher fluorescence was found in Se-treated roots of A. membranaceus compared with the control, while it diminished after Se application in A. bisulcatus (Fig. 4D). Lignin and suberin deposition was visualized using Auramine O staining in the root sections. An intense fluorescence was found in the stele in control roots of both species due to the xylem vessels (Fig. 4E). In the Se-treated roots of A. membranaceus, a slight fluorescence appeared on the surface (exodermis) of the roots. This staining affected both the endodermis and the exodermis in the roots of Se-treated A. bisulcatus. Fig. 4 View largeDownload slide Root diameter (A), the thickness of the cortex (B) and the diameter of the stele in the roots (C) of control (Cont) and 50 or 100 µM selenate-treated (50 Se and 100 Se) Astragalus species after 14 d. The values of aniline blue (AB) fluorescence (pixel intensity) which refers to callose deposition are given in Control% (D). Auramine O staining of the control and Se-treated root sections of both species (E). Strong fluorescence can be seen at the xylem vessels (white arrows) and the endodermis and/or the exodermis (red arrows). Scale bar = 100 µm. Different letters refer to significant differences among the treatments within the same species according to Kruskal–Wallis ANOVA at P < 0.05 (n = 6). Significant differences between the species within the same treatment were determined by Mann–Whitney U-test and are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant). Fig. 4 View largeDownload slide Root diameter (A), the thickness of the cortex (B) and the diameter of the stele in the roots (C) of control (Cont) and 50 or 100 µM selenate-treated (50 Se and 100 Se) Astragalus species after 14 d. The values of aniline blue (AB) fluorescence (pixel intensity) which refers to callose deposition are given in Control% (D). Auramine O staining of the control and Se-treated root sections of both species (E). Strong fluorescence can be seen at the xylem vessels (white arrows) and the endodermis and/or the exodermis (red arrows). Scale bar = 100 µm. Different letters refer to significant differences among the treatments within the same species according to Kruskal–Wallis ANOVA at P < 0.05 (n = 6). Significant differences between the species within the same treatment were determined by Mann–Whitney U-test and are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant). Se-induced changes in ROS and RNS metabolism in root and shoot tissues One possible molecular mechanism of Se phytotoxicity is the production of ROS and the consequent oxidative stress (Van Hoewyk 2013). Recently, Se-triggered nitrosative processes have also been discovered, which are caused by the disturbance of RNS metabolism (Kolbert et al. 2016). The ROS- and RNS-inducing effects of Se were compared in the organs of Astragalus species (Fig. 5) in order to reveal the possible link between Se tolerance or sensitivity and Se-induced oxidative and nitrosative (together with nitro-oxidative) stress. Fig. 5 View largeDownload slide (A) The level of superoxide in the root tips of A. membranaceus and A. bisulcatus treated with 0, 50 or 100 µM selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Representative fluorescent microscopic images showing DHE-stained root tips of Astragalus species. Scale bars = 500 µm. (C) Representative photographs taken from NBT-stained cotyledons of control (0 Se), 50 or 100 µM selenate-treated A. membranaceus and A. bisulcatus. The blue discoloration refers to superoxide accumulation. Scale bar = 1 cm. (D) Native-PAGE (10%) separation of NOX isoenzymes in cotyledon and root of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. The most representative protein band is indicated as ‘main band’. Additional putative isoenzymes are indicated by black arrows, and newly appeared NOX isoenzymes are labeled by asterisks. (E and F) Total activity of SOD enzymes in the organs of Astragalus species supplemented (50 or 100 µM) or not (0 µM) with selenate. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). (G) Native-PAGE separation (10%) of SOD isoenzymes in cotyledon and root of control and selenate-treated Astragalus species. Fig. 5 View largeDownload slide (A) The level of superoxide in the root tips of A. membranaceus and A. bisulcatus treated with 0, 50 or 100 µM selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Representative fluorescent microscopic images showing DHE-stained root tips of Astragalus species. Scale bars = 500 µm. (C) Representative photographs taken from NBT-stained cotyledons of control (0 Se), 50 or 100 µM selenate-treated A. membranaceus and A. bisulcatus. The blue discoloration refers to superoxide accumulation. Scale bar = 1 cm. (D) Native-PAGE (10%) separation of NOX isoenzymes in cotyledon and root of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. The most representative protein band is indicated as ‘main band’. Additional putative isoenzymes are indicated by black arrows, and newly appeared NOX isoenzymes are labeled by asterisks. (E and F) Total activity of SOD enzymes in the organs of Astragalus species supplemented (50 or 100 µM) or not (0 µM) with selenate. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). (G) Native-PAGE separation (10%) of SOD isoenzymes in cotyledon and root of control and selenate-treated Astragalus species. In root tips of A. membranaceus, both Se concentrations increased superoxide levels, although the highest and significant superoxide production was observed in the case of 50 µM Se, resulting in a 170% increase (Fig. 5A, B). In the root tips of tolerant A. bisulcatus, selenate had no effect on superoxide levels (Fig. 5A, B). In intact cotyledons, superoxide levels were examined qualitatively by nitroblue tetrazolium (NBT) staining (Fig. 5C). In the case of 50 or 100 µM Se-treated A. membranaceus plants, the intense presence of blue colorization indicated superoxide production. In A. bisulcatus, slightly intensified blue staining was detected only as the effect of 100 µM Se treatment (Fig. 5C). In order to reveal the mechanism of the different superoxide responses of the species, we examined the metabolism of this reactive intermediate. The superoxide-generating NADPH oxidase (NOX) isoenzymes were separated by native-PAGE and a protein band which was strongly present in all samples was determined (Fig. 5D ‘main band’). In the cotyledons of A. bisulcatus one, while in A. membranaceus four additional putative NOX isoenzymes were detected. As the effect of Se, only slight changes occurred in NOX isoenzyme activities especially in A. bisulcatus, while more protein bands showed increased activity in A. membranaceus cotyledons (Fig. 5D;Supplementary Fig. S3). In the roots of both species, the activity of the main NOX protein band was less pronounced, although Se induced its activity in A. bisulcatus roots. In addition to the main protein band, four other isoenzymes were detected in A. bisulcatus roots, three of which showed induction as the effect of selenate exposure (Fig. 5D;Supplementary Fig. S3). In the case of A. membranaceus roots, Se reduced the activity of the main NOX band, which seemed to be substituted by the appearance and strong activation of additional, putative NOX isoenzymes (indicated by asterisks in Fig. 5D and Supplementary Fig. S3). Both concentrations of selenate caused notable (∼30% and 38%) induction of superoxide-eliminating SOD enzymes in A. membranaceus roots, while the effect of selenate in the root system of A. bisulcatus proved to be much slighter (∼10%, Fig. 5E). Regarding the cotyledons, selenate exposure resulted in SOD activation only in A. membranaceus, and the effect proved to be slighter compared with the root (∼15%, Fig. 5F). We separated SOD isoforms by native-PAGE, and differences were observed between the species and also between the organs (Fig. 5E). In both organs of A. bisulcatus, four activity bands (Mn SODI, Fe SOD I, Fe SOD II and Cu/Zn SOD) were identified, while in A. membranaceus cotyledons six bands were detected (Mn SODII, Fe SOD I, Fe SOD II, Cu/Zn SOD I, Cu/Zn SOD II and Cu/Zn SOD III). Moreover, in the root system of A. membranaceus, only three SOD activity bands (Mn SODII, Fe SODI and Fe SODII) were observed. Quantification showed that selenate at the higest applied concentration exerted a slight effect on SOD isoenzymes in A. bisulcatus cotyledons (Supplementary Fig. S4). In contrast, five SOD isoforms out of six showed intensified activity as the effect of 50 µM Se in cotyledons of A. membranaceus. Regarding the root system, both applied Se treatments induced the activity of Mn, Fe and Cu/Zn SODs in A. bisulcatus, but these inductions were much more intense in A. membranaceus (Supplementary Fig. S4). Similar to superoxide, NO formation was significantly enhanced as an effect of 50 µM Se in the root of sensitive A. membranaceus (Fig. 6A, B). Regarding peroxynitrite, 50 µM Se resulted in its accumulation, but the highest Se dose decreased its level in the root tips of the sensitive species (Fig. 6E, F). Interestingly, none of the applied Se treatments had any observable effect on the examined RNS levels in A. bisulcatus root tips (Fig. 6A, B, E, F). Fig. 6 View largeDownload slide The level of nitric oxide (A–D) and peroxynitrite (E–H) in intact root tips (A, B, E, F) and cotyledon cross-sections (C, D, G, H) of control (0 µM), 50 µM or 100 µM selenate-treated A. membranaceus and A. bisulcatus. Scale bars = 500 µm. (I–L) Immunofluorescent detection of GSNO in cross-sections of roots (I and J) and cotyledons (K and L). Scale bars = 200 µm. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). (M) Native-PAGE (6%) of Astragalus cotyledon and root extracts and staining for GSNOR activity. Astragalus membranaceus and A. bisulcatus were treated with 0, 50 or 100 µM selenate for 14 d. Fig. 6 View largeDownload slide The level of nitric oxide (A–D) and peroxynitrite (E–H) in intact root tips (A, B, E, F) and cotyledon cross-sections (C, D, G, H) of control (0 µM), 50 µM or 100 µM selenate-treated A. membranaceus and A. bisulcatus. Scale bars = 500 µm. (I–L) Immunofluorescent detection of GSNO in cross-sections of roots (I and J) and cotyledons (K and L). Scale bars = 200 µm. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). (M) Native-PAGE (6%) of Astragalus cotyledon and root extracts and staining for GSNOR activity. Astragalus membranaceus and A. bisulcatus were treated with 0, 50 or 100 µM selenate for 14 d. Unlike the roots, both species showed NO accumulation in their cotyledons as the effect of 50 µM Se (Fig. 6C, D). Both Se concentrations triggered significant peroxynitrite generation in the cotyledons of A. membranaceus, while in the tolerant species only slight, non-significant changes were observed (Fig. 6G, H). Se-induced alterations in GSNO levels were also determined in the root and shoot tissues of the species (Fig. 6I–L). Under control conditions, significantly higher GSNO content was determined in both organs of A. bisulcatus compared with A. membranaceus. Selenate treatments caused significant reduction in GSNO levels of both A. bisulcatus organs. A similar Se-induced diminution of GSNO content was found in A. membranaceus roots (Fig. 6I, J); however, in the cotyledons, Se exposure led to a significant and concentration-dependent increase of GSNO levels (Fig. 6K, L). Significantly increased fluorescence was detected in GSNO-pre-treated sections, which served as positive controls, while light-inactivated GSNO did not result in an increase in fluorescence (Supplementary Fig. S7). In their cotyledons, both species showed relatively high S-nitrosoglutathione reductase (GSNOR) activity compared with the root system during control conditions (Fig. 6M;Supplementary Fig. S6). Selenate exerted an inhibitory effect on GSNOR activity in A. bisulcatus cotyledons, while it notably induced it in the cotyledons of 50 µM selenate-treated A. membranaceus. As for the control root system, A. bisulcatus showed higher GSNOR activity than A. membranaceus where the activity was barely detectable (Fig. 6M;Supplementary Fig. S6). In the case of A. bisulcatus, selenate exerted a concentration-dependent reducing effect on GSNOR activity. In contrast to this, selenate did not modify the enzyme activity in the root of A. membranaceus (Fig. 6M;Supplementary Fig. S6). Selenium-induced protein tyrosine nitration Protein tyrosine nitration as a consequence of RNS accumulation was investigated by both immunofluorescence (Fig. 7) and Western blot analysis (Fig. 8). In cross-sections of A. membranaceus primary roots, an immunofluorescent signal related to 3-nitrotyrosine was observable mainly in the endodermal cell layer and within the central cylinder (Fig. 7B). Se exposure led to a significant increase in the 3-nitrotyrosine-dependent fluorescent signal in all tissues of the root (Fig. 7A), but this elevation was the most pronounced in the central cylinder (Fig. 7B). Under control conditions, 3-nitrotyrosine was located mainly in the endodermal cell layer of A. bisulcatus roots (Fig. 7B). Milder Se treatment caused a slight increase of the fluorescence in the endodermis, and the most serious Se exposure induced 3-nitrotyrosine accumulation in all tissues of the primary root, although this increase was smaller than in A. membranaceus roots (Fig. 7A). In cotyledons, Astragalus species showed differences in physiological 3-nitrotyrosine levels, since A. bisulcatus showed higher 3-nitrotyrosine-related fluorescence (Fig. 7C). Moreover, high levels of 3-nitrotyrosine were found to be located in cotyledon veins (Fig. 7D). Both selenate treatments significantly decreased the 3-nitrotyrosine content of A. bisulcatus cotyledons, but in the case of A. membranaceus, 100 µM selenate induced 3-nitrotyrosine formation (Fig. 7C). As positive and negative controls, sections were treated with SIN-1, and enhanced fluorescence intensity was detected, while urate pre-treatment remarkably mitigated 3-nitrotyrosine-dependent fluorescence (Supplementary Fig. S7). Fig. 7 View largeDownload slide The intensity of 3-nitrotyrosine-related fluorescence in root (A) or cotyledon (C) cross-sections of control and selenate-treated A. membranaceus and A. bisulcatus. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). Representative fluorescent microscopic images showing cross-sections of roots (B) and cotyledons (D) of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. Scale bars = 200 or 500 µm. Fig. 7 View largeDownload slide The intensity of 3-nitrotyrosine-related fluorescence in root (A) or cotyledon (C) cross-sections of control and selenate-treated A. membranaceus and A. bisulcatus. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). Representative fluorescent microscopic images showing cross-sections of roots (B) and cotyledons (D) of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. Scale bars = 200 or 500 µm. Fig. 8 View largeDownload slide Protein and tyrosine nitration pattern in cotyledon and root of control and selenate-treated Astragalus species (25 µg per lane). Silver-stained SDS gels (12%) and Western blots probed with a rabbit anti-nitrotyrosine polyclonal antibody (1:2,000). Commercial nitrated BSA (NO2-BSA) was used as a positive control, and the molecular marker is shown as a protein weight indicator. Gray arrows indicate intensification of nitration, and white arrows show protein bands with decreased nitration. Selenate-induced, newly appeared protein bands are indicated by black arrows. Fig. 8 View largeDownload slide Protein and tyrosine nitration pattern in cotyledon and root of control and selenate-treated Astragalus species (25 µg per lane). Silver-stained SDS gels (12%) and Western blots probed with a rabbit anti-nitrotyrosine polyclonal antibody (1:2,000). Commercial nitrated BSA (NO2-BSA) was used as a positive control, and the molecular marker is shown as a protein weight indicator. Gray arrows indicate intensification of nitration, and white arrows show protein bands with decreased nitration. Selenate-induced, newly appeared protein bands are indicated by black arrows. In the whole protein extract, tyrosine nitration was determined by Western blot analysis (Fig. 8). In A. membranaceus cotyledons, selenate intensified tyrosine nitration of five protein bands (∼27, 22, 17, 12 and 10 kDa, indicated by gray arrows) but a newly nitrated protein band could not be observed. In cotyledons of A. bisulcatus, both Se treatments resulted in the appearance of a highly nitrated protein band (with high molecular weight, indicated by black arrows) but Se did not cause any other nitration-related change in the proteome. In A. bisulcatus roots, Se did not intensify protein tyrosine nitration, and even caused a decrease in three protein bands (∼75, 12 and 10 kDa). In contrast, the Se-sensitive Astragalus species showed several protein bands whose immunopositivity towards anti-3-nitrotyrosine showed an Se-dependent appearance. Discussion Both species were able to take up selenate from the external media (Fig. 1). Even though A. membranaceus accumulated a large amount of Se in its root, the root to shoot Se translocation proved to be slight. In contrast, in A. bisulcatus cotyledons, >7-fold Se concentrations were measured compared with A. membranaceus, indicating a high rate of Se translocation. Indeed, the root to shoot Se ratio was 3.8 in A. bisulcatus plants grown on 100 µM selenate, suggesting that it is a hyperacumulator species (Freeman et al. 2010). Furthermore, the relatively high endogenous Se content in the organs of control A. bisulcatus indicates its hyperaccumulator nature. Also the amount of the accumulated Se (∼1,800 µg g–1 DW in the cotyledons of 100 µM selenate-exposed plants) supports the hyperaccumulation capability of A. bisulcatus (Mehdawi and Pilon-Smits 2012). In addition to Se, exogenous selenate affected the concentrations of essential microelements such as Fe, Zn, Mn and B (Table 1) especially in A. membranaceus, inhibiting their absorption and consequently causing disturbances in their homeostasis. Similar antagonism between Se and macro- or microelements has earlier been described by others (Pazurkiewicz-Kocot et al. 2003, Filek et al. 2010, Zembala et al. 2010). Reduced availability of essential microelements may worsen the growth and physical condition of the plant. B is needed to maintain cell wall integrity, while Zn protects membrane lipids and proteins, and, together with Mn, Cu and Fe, is the metal component of SOD antioxidant enzymes (Cakmak 2000). In the case of the Se hyperaccumulator A. bisulcatus, the microelement homeostasis seems to be more stable, since Se did not cause disturbance in it, which may contribute to the better tolerance of this species. Se negatively affected the germination capability and the biomass production of young A. membranaceus, but the germination and growth of A. bisulcatus proved to be insensitive to Se (Fig. 2). However, root elongation concentration dependently decreased as the effect of elevated Se concentrations, suggesting the higher sensitivity of the root system to Se compared with the aerial plant parts (Lehotai et al. 2016). Because of the Se concentration-dependent response of elongation, root growth can be used as an indicator of Se tolerance (Molnár et al. 2018a, Tamaoki et al. 2008). The hyperaccumulator A. bisulcatus was able to maintain its root growth on Se-containing medium (Fig. 3A) even though meristem cells suffered a certain degree of loss of viability (Fig. 3B, C). The reduced root elongation (Fig. 3A) and meristem viability (Fig. 3B, C) of A. membranaceus indicate its sensitivity to Se. Beyond the viability of the root apical meristem, in the background of Se-inhibited organ development, the disturbances of hormone homeostasis or unfavorable alterations in primary metabolism can also be determined (reviewed by Kolbert et al. 2016). Based on the observed parameters (germination, biomass production, root elongation and cell viability), young A. membranaceus proved to be Se sensitive, while the hyperaccumulator A. bisulcatus showed remarkable Se tolerance, which supports the previously described connection between Se hyperaccumulation and (hyper)tolerance (Mehdawi and Pilon-Smits 2012). The main reason for Se tolerance of A. bisulcatus is that this species expresses the SMT enzyme which prevent toxic seleno-amino acid formation (Neuhierl and Bock 1996). Considering the high shoot Se accumulation (Fig. 1B), it can be assumed that the notable Se tolerance of A. bisulcatus is due to detoxification and not exclusion. We observed Se-induced alterations in root structure of both Astragalus species. Thicker roots of control and 50 µM Se-treated sensitive A. membranaceus compared with A. bisulcatus were probably due to the thicker cortex (Fig. 4A, B). The increment of the root diameter, including the thickening of the cortex is common in heavy metal-stressed plant roots (Arduini et al. 1995, Maksimović et al. 2007, Potters et al. 2007). The hyperaccumulator species A. bisulcatus showed more intense Se-induced root thickening than A. membranaceus (Fig. 4A–C), which is in agreement with the results of Li et al. (2009) where in the hyperaccumulating ecotype of Sedum alfredii, a lead/zinc-triggered increment in root diameter and other root morphological parameters was observed. The deposition of callose seems to be a good marker of stress-induced cell wall alterations. It was formerly found that copper can induce callose formation in onion epidermal cells and in the root tips of Brassica species (Kartusch 2003, Feigl et al. 2013). In our study, the sensitive Astragalus species showed both Se-triggered callose accumulation (Fig. 4D) and exodermal suberin lamellae deposition (Fig. 4E; Dalla Vecchia et al. 1999, Rahoui et al. 2017) which together may serve as an extracellular barrier limiting water and mineral uptake. This may result in Se exclusion and at the same time the inhibition of growth. In the case of A. bisulcatus, not only the exodermis but also the endodermis exhibited the presence of suberin (Fig. 4E). Since exodermal suberin deposition occurs earlier in time followed by the appearance of endodermal suberin as the effect of metal stress (Vaculík et al. 2012), we can conclude that in the case of A. membranaceus, the delayed formation of Se-induced endodermal suberin lamellae is associated with Se sensitivity. Moreover, the development of apoplastic barriers (exodermal and endodermal) can be considered as an adaptive trait (Vaculík et al. 2012). For the toxic effect of Se, the accumulation of ROS and the consequent oxidative stress is partly responsible (Van Hoewyk 2013). The accumulation of the rapidly generating, harmful ROS, superoxide anion (Fig. 5A–C), as well as the induction of SOD activity (Fig. 5E, F) suggest Se-triggered oxidative stress in A. membranaceus organs, while no sign of serious oxidative damage was observed in A. bisulcatus. The expression of superoxide-generating NOX isoenzymes showed species specificity in A. membranaceus roots, and newly expressed NOX isoenzymes were observed as the effect of selenate (Fig. 5D). Regarding SOD isoenzymes, A. membranaceus cotyledons express more Cu/Zn SODs than A. bisulcatus, and selenate remarkably increased the activity of most of the isoenzymes (Fig. 5G). Se-triggered superoxide accumulation has been observed in the non-accumulatord Stanleya albescens and Arabidopsis thaliana and in secondary accumulators such as Brassica napus, Brassica rapa and Brassica juncea (Molnár et al. 2018a, Molnár et al. 2018b, Tamaoki et al. 2008, Freeman et al. 2010, Chen et al. 2014, Dimkovikj and Van Hoewyk 2014). In the hyperaccumulator species Stanleya pinnata, elevated levels of ROS-scavenging compounds (ascorbate and GSH) were observed which are involved in the prevention of Se-induced oxidative stress (Freeman et al. 2010). In our study, A. bisulcatus showed moderately higher SOD activities (especially Cu/Zn SODs) in the roots compared with A. membranaceus (Fig. 5F) which may contribute to endurance against Se-induced oxidative stress. At the same time, Se hyperaccumulators are known to accumulate organic Se forms (mainly methyl-seleno-cysteine) instead of the oxidative stress-inducing inorganic Se compounds, which may be a relevant protection mechanism against oxidative stress (Schiavon and Pilon-Smits 2017). Additionally, Se exposure has been shown earlier to disturb the metabolism of RNS. A milder selenate dose triggered NO production mainly in the non-accumulator species (Fig. 6A–D) similarly to selenite-exposed Pisum sativum (Lehotai et al. 2016) or the selenate-treated secondary accumulator B. rapa (Chen et al. 2014). Based on the results of Rios et al. (2010), it is conceivable that selenate induces nitrate reductase (NR) which is the main enzymatic NO source in the root system and is also involved in NO production in the aerial plant parts (Zhang et al. 2011). The effect of Se on NR activity can be direct or indirect since Se-induced S deficiency may increase Mo content, thus inducing NR (Shinmachi et al. 2010, Yu et al. 2010). In our experiments, significantly higher Mo concentrations were measured in both organs of selenate-treated A. membranaceus (Table 1) which can be connected to the elevated NO production. Peroxynitrite can be formed in vivo in the fast reaction between superoxide radical and NO (Kissner et al. 1997), thus their accumulation may predict and explain Se-induced ONOO– generation. The concentration of this strong oxidative and nitrosative agent could reflect overall stress severity (Arasimowicz-Jelonek and Floryszak-Wieczorek 2011); therefore, we can suspect that A. membranaceus suffers more severe Se-triggered nitro-oxidative stress compared with A. bisulcatus. However, like the effect of the highest Se dose in A. membranaceus root, the peroxynitrite level decreases (Fig. 6E) due to the possible activation of scavenging mechanisms. GSNO is a mobile form of NO storage in plants, being responsible for protein S-nitrosylation. The spontaneous decomposition of GSNO leads to NO production, while it is enzymatically reduced by GSNOR or it can catalyze the transnitrosylation of protein thiols leading to its decomposition (Begara-Morales et al. 2018, Lindermayr 2018). Both species responded to the presence of selenate by decreasing the endogenous GSNO reservoir of their roots (Fig. 6I); however, this resulted in NO accumulation only in A. membranaceus (Fig. 6A). Presumably, in A. bisulcatus the originally high GSNO content participated in transnitrosylation reactions with cysteine thiols in proteins leading to S-nitrosothiol (SNO) formation, and GSNOR-catalyzed reduction is not involved in GSNO metabolism under Se stress. In the cotyledon of A. bisulcatus, the level of GSNO decreased (Fig. 6K, L) possibly due to spontaneous decomposition yielding NO but not GSNOR activity. Similarly to other species (reviewed by Corpas et al. 2013), both A. membranaceus and A. bisulcatus can be characterized by a certain physiological nitropoteome which means that a part of their protein pool is nitrated even in the control state. Both the Se-induced increase in fluorescence intensity (Fig. 7), and the presence of several newly nitrated protein bands (Fig. 8) indicated more intense protein tyrosine nitration in the organs of A. membranaceus compared with the hyperaccumulator A. bisulcatus. Moreover, both immunofluorescence and Western blot results showed that the tolerant species possesses a large physiological nitroproteome as well as large mobile NO storage form (GSNO) with which it is able to buffer NO radical content. The Se-induced GSNO and 3-nitrotyrosine decompositions without the accumulation of the reactive ·NO may contribute to tolerance against nitro-oxidative stress in A. bisulcatus. The Se-triggered decrease in the amount of 3-nitrotyrosine may be conceivable via proteasomal degradation (Castillo et al. 2015). Our experiments examined the sensitivity of young non-accumulator A. membranaceus and hyperaccumulator A. bisulcatus to Se in connection to secondary oxidative and nitrosative processes, and the obtained results are summarized in Fig. 9. As expected, the observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth and cell viability) indicated that A. membranaceus is Se sensitive while A. bisulcatus tolerates the presence of high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism involving NOXs and SODs, leading to superoxide accumulation. Furthermore, this study points out for the first time that Se induced the production or disturbed the metabolism of RNS (NO, ONOO– and GSNO) consequently resulting in intensified protein tyrosine nitration in the sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se decreased the high GSNO content and tyrosine nitroproteome without the accumulation of the NO radical, resulting in the lack of tyrosine nitration. These findings suggest that this species is able to prevent Se-induced nitro-oxidative stress to which enhanced ROS/RNS-scavenging capability may also contribute. Given that the elevated levels of other elements (e.g. zinc, arsenic and cadmium) have been reported to induce protein nitration and cause similar disturbances in ROS and RNS metabolism to Se (Leterrier et al. 2012, Feigl et al. 2015, Feigl et al. 2016, Liu et al. 2018), excess Se-induced nitro-oxidative stress can be considered a general rather than an Se-specific phenomenon. Future research should focus on the evaluation of the antioxidative system in order to obtain a more accurate view of nitro-oxidative processes in relation to Se tolerance. Fig. 9 View largeDownload slide Schematic model summarizing the data obtained in this study. In the sensitive species, selenium exposure induces intense modification of root cell wall structure, disturbs microelement homeostasis and induces NO, superoxide and peroxynitrite accumulation as well as protein tyrosine nitration (nitro-oxidative stress). The observed alterations together lead to selenium-induced damage. In contrast, the Se-tolerant species shows slight cell wall modifications and non-disturbed microelement homeostasis. Additionally, selenium does not trigger NO, superoxide, peroxynitrite or 3-nitrotyrosine formation; instead the high amount of endogenous NO storage (GSNO) and the large nitroproteome decrease without the accumulation of NO, suggesting that GSNO and/or the nitrosoproteome are able to buffer the amount of NO radical. In the hyperaccumulator, slight Se-triggered damage or the complete lack of damage can be observed. See details in the text. Abbreviations: 3NT = 3-nitrotyrosine. Fig. 9 View largeDownload slide Schematic model summarizing the data obtained in this study. In the sensitive species, selenium exposure induces intense modification of root cell wall structure, disturbs microelement homeostasis and induces NO, superoxide and peroxynitrite accumulation as well as protein tyrosine nitration (nitro-oxidative stress). The observed alterations together lead to selenium-induced damage. In contrast, the Se-tolerant species shows slight cell wall modifications and non-disturbed microelement homeostasis. Additionally, selenium does not trigger NO, superoxide, peroxynitrite or 3-nitrotyrosine formation; instead the high amount of endogenous NO storage (GSNO) and the large nitroproteome decrease without the accumulation of NO, suggesting that GSNO and/or the nitrosoproteome are able to buffer the amount of NO radical. In the hyperaccumulator, slight Se-triggered damage or the complete lack of damage can be observed. See details in the text. Abbreviations: 3NT = 3-nitrotyrosine. Materials and Methods Plant material and growing conditions Astragalus bisulcatus (Hook.) A. Gray seeds were obtained from B&T World Seeds (Aigues-Vives, France), and Astragalus membranaceus (Fisch.) Bunge seeds were provided by Professor Aaron Chang (Kaohsiung Medical University, Graduate Institute of Natural Products, Kaohsiung, Taiwan). Seeds were surface sterilized with 20% (v/v) sodium hypochlorite for 20 min, and washed with sterile distilled water four times in 20 min. Seeds were dried on a sterile metal filter and we polished them one by one using P-400 sanding paper in order to scratch the external seed coat. Seeds were placed on agar medium (the scratched surface of the seeds contacted the medium). Plastic, square Petri dishes contained half-strength Murashige and Skoog medium [0.8% (v/v) agar, 1% sucrose] supplemented with 0 (control), 50 or 100 μM sodium selenate (Na2SeO4). Both plant species were grown under controlled conditions (150 µmol m– s–1 photon flux density, 12 h/12 h light/dark cycle, relative humidity 55–60% and temperature 25 ± 2°C) for 14 d. All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Se and microelement content analysis Cotyledon and root materials of both Astragalus species were harvested separately and rinsed with distilled water then dried at 70°C for 72 h. Nitric acid [65% (w/v), Reanal] and H2O2 [30% (w/v) VWR Chemicals] were added to dried plant material. The samples were destroyed in a microwave destructor (MarsX-press CEM) at 200°C and 1,600 W for 15 min. After appropriate dilutions with distilled water, the samples were transferred to 20 ml Packard glasses. Element concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700 Series). Concentrations of Se and essential microelements (Fe, Zn, Mn, Mo are B) are given in µg g–1 DW. These analyses were carried out twice with three samples each (n = 3). Evaluation of germination, growth parameters, root cell viability and Se tolerance index Germinated seeds were counted in each Petri dish and germination percentages (%) were calculated. Fresh weights of root and shoot materials were measured using a balance, and the values are given in milligrams. Lengths of primary roots were measured manually. From the data, the Se tolerance index (%) was calculated according to the following formula: tolerance index (%) = (treated root length/mean control root length)×100 Cell viability in the root apical meristem was determined by using the FDA fluorophore. Root tips were incubated in 10 µM FDA solution (prepared in 10/50 mM MES/KCl buffer, pH 6.15) for 30 min in darkness and were washed four times in buffer. These data were acquired from three separate generations, and in each generation 15 plants/seeds were examined (n = 15). Evaluation of tissue-level changes in the roots induced by selenium Small pieces of root samples derived from the mature zone were fixed in 4% (w/v) paraformaldehyde according to Barroso et al. (2006). After the fixation, root samples were washed in distilled water and embedded in 5% agar (bacterial; Zelko et al. 2012 with modifications). Then 100 µm thick cross-sections were prepared using a vibratome (VT 1000S, Leica). The sections were placed on a slide with a drop of water and were stained with aniline blue [0.5% (w/v)] to detect the deposition of callose. The root sections were observed by a light microscope and an inverted fluorescent microscope (Zeiss Axiovert 200 M, Carl Zeiss) equipped with a digital camera (AxiocamHR, HQ CCD, Carl Zeiss). Images obtained by light microscopy were applied to measure several parameters of the root such as root diameter, the thickness of the cortex and the diameter of the stele according to Arduini et al. (1995). All data are given in micrometers. Fluorescent microscopy was applied to observe the fluorescence of secondary cell wall compounds such as lignin and suberin (Auramine O staining) as well as the formation of callose as a result of Se stress, using filter set 9 (excitation, 450–490 nm; emission, 515–∞ nm) and filter set 49 (excitation, 365 nm; emission 445/50 nm) (Feigl et al. 2013, Rahoui et al. 2017). In both cases, fluorescence intensity (pixel intensity) was measured on digital images applying Axiovision Rel. 4.8 software (Carl Zeiss) within circles of 100 µm radius which were set to cover the largest area of the vascular cylinder. The data of the Se-treated plants were calculated as a percentage of the control. These experiments were carried out on two separate plant generations with six plants examined in each (n = 6). In situ detection of ROS and RNS in the root tips and in cotyledons Dihydroethidium (DHE) at 10 µM concentration was applied for the detection of superoxide anion levels in the roots. Root segments were incubated for 30 min in darkness at 37°C, and washed twice with Tris–HCl buffer (10 mM, pH 7.4) (Kolbert et al. 2012). In cotyledons, instead of DHE, NBT was used for visualizing superoxide production. Excised cotyledons were incubated in Falcon tubes containing 5 ml of NBT solution (1 mg ml–1 in 10 mM phosphate buffer, pH 7.4) for 30 min under illumination. Pigments were removed by incubating the cotyledons in 80% (v/v) ethanol at 70°C for 30 min. The NO level of the root tips and in handmade cross-sections from cotyledons was monitored with the help of 4-amino-5-methylamino- 2′,7′-difluorofluorescein diacetate (DAF-FM DA) according to Kolbert et al. (2012). Root and cotyledon segments were incubated in 10 µM dye solution for 30 min (darkness, 25 ± 2°C), and washed twice with Tris–HCl (10 mM, pH 7.4). Peroxynitrite was also visualized in root tips and in handmade cross-sections of cotyledons. Samples were incubated in 10 µM dihydrorhodamine 123 (DHR) prepared in Tris–HCl buffer. After 30 min of incubation at room temperature, root tips and cotyledon segments were washed twice with the buffer solution (Sarkar et al. 2014). These analyses were carried out twice with 10 samples each (n = 10). Determining SOD, NADPH oxidase izoenzymes and GSNOR activity by native-PAGE Fresh cotyledon and root tissues of A. bisulcatus and A. membranaceus were ground with a double volume of extraction buffer (50 mM Tris–HCl buffer pH 7.6–7.8) containing 0.1 mM EDTA, 0.1% Triton X-100 and 10% glycerol, and centrifuged at 12,000 r.p.m. for 20 min at 4°C. The protein extract was treated with 1% protease inhibitor cocktail and stored at –20°C. Protein concentration was determined using the Bradford (1976) assay with bovine serum albumin (BSA) as a standard. In order to avoid the effect of the changes in protein concentration and composition induced by the treatments, our data are standardized to fresh weight by loading equal volumes of protein extracts in each well. Silver staining was performed according to Blum et al. (1987) with slight modifications. The gel was fixed with methanol and acetic acid, then treated with a sensitizing solution and staining solution containing AgNO3. The gel was developed in a solution containing sodium carbonate and formaldehyde (Supplementary Figs. S2, S5). NOX activity was examined on 10% native polyacrylamide gels by the NBT reduction method of López-Huertas et al. (1999) with slight modifications. In the case of cotyledons 15 µl and in the case of roots 25 µl of protein extracts were loaded in each well. Following electrophoresis, the gel was incubated in reaction buffer (50 mM Tris–HCl pH 7.4, 0.1 mM MgCl2, 1 mM CaCl2) containing 0.2 mM NBT and 0.2 mM NADPH for 20 min in darkness. As a positive control, the NOX-specific inhibitor diphenylene iodonium (DPI) was used at a final concentration of 50 µM. In addition, NADPH-independent superoxide production was examined on a gel without NAPDH supplementation. SOD activity was measured based on the ability of the enzyme to inhibit photochemical reduction of NBT catalyzed by riboflavin, as described by Dhindsa et al. (1981). A 250 mg aliquot of plant biomass was ground with 10 mg of polyvinyl polypyrrolidone (PVPP) in 1 ml of 50 mM pH 7.0 phosphate buffer containing 1 mM EDTA. The enzyme activity is expressed as specific activity (U g–1 FW weight), where 1 unit of SOD activity means 50% inhibition of NBT reduction in light. For the examination of SOD activity and isoenzymes, protein extracts (15 and 25 µl in the case of cotyledons and roots, respectively) were subjected to native gel electrophoresis on a 10% polyacrylamide gel (Beauchamp and Fridovich 1971). The gel was rinsed in 50 mM potassium phosphate buffer (pH 7.8) twice, ncubated for 20 min in 2.45 mM NBT in darkness and then for 15 min in freshly prepared 28 mM TEMED solution containing 2.92 µM riboflavin. After the incubation, the gels were washed twice and developed by light exposure. SOD isoforms were identified by incubating gels in 50 mM potassium phosphate containing 2 mM potassium cyanide to inhibit Cu/Zn SOD activity, or 5 mM H2O2 which inhibits Cu/Zn and Fe SOD activity for 30 min before staining with NBT. Mn SODs are resistant to both inhibitors. GSNOR activity was visualized using a slightly modified method of that by Seymour and Lazarus (1989). Native-PAGE was performed using 6% acrylamide gels with Tris-boric-EDTA buffer (8.9 mM Tris base, 8.9 mM boric acid and 0.2 mM Na2EDTA, pH 8). In the case of cotyledons 30 µl, and in case of roots 50 µl of protein extracts were loaded in each well. Gels were incubated for 15 min at 4°C in the presence of 2 mM NADH solution prepared in 100 mM sodium phosphate buffer (pH 7.4). Excess buffer was removed and a filter paper containing freshly prepared 3 mM GSNO solution (prepared in 100 mM sodium phosphate buffer, pH 7.4) was added (15 min, darkness, 4°C). NADH UV fluorescence was visualized at 312 nm wavelength using a gel documentation system (Image System Felix 1000/2000, Biostep). GSNOR enzyme activity consumed NADH, resulting in dark bands in the gel. Relevant bands showing NOX, SOD or GSNOR signals were quantified by Gelquant software (provided by biochemlabsolutions.com) and the data are presented as Supplementary Figs. S3, S4 and S6, respectively. These experiments were carried out on two separate plant generations with three samples examined each (n = 3). Immunofluorescent detection of GSNO and 3-nitrotyrosine in root and cotyledon cross-sections Cross-sections were prepared using a vibratome as described earlier, and immunodetection was performed according to Corpas et al. (2008) with slight modifications. Free-floating sections were incubated at room temperature overnight with rat antibody against GSNO (VWR Chemicals) diluted 1:2,500 in TBSA–BSAT solution containing 5 mM Tris buffer (pH 7.2), 0.9% (w/v) NaCl, 0.05% (w/v) sodium azide, 0.1% (w/v) BSA and 0.1% (v/v) Triton X-100. Samples were washed three times with TBSA–BSAT solution within 15 min. After the washings, cross-sections were incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-rat IgG secondary antibody (1:1,000 in TBSA–BSAT, Agrisera) for 1 h at room temperature. Samples were placed on microscopic slides in phosphate-buffered saline (PBS):glycerine (1:1). As a positive control, cross-sections were treated with 250 μM GSNO (prepared in TBSA–BSAT) for 1 h prior to the labeling process. Light-inactivated GSNO was prepared as described by Wodala and Horváth (2008) and was applied for 1 h prior to labeling. Immunodetection of 3-nitrotyrosine was carried out according to Valderrama et al. (2007). Samples were incubated for 3 d at 4°C with polyclonal rabbit antibody against 3-nitrotyrosine (Sigma-Aldrich) diluted in TBSA–BSAT (1:300). After three washes with TBSA–BSAT, sections were incubated for 1 h at room temperature in FITC-conjugated goat anti-rabbit IgG (1:1,000 in TBSA––BSAT, Agrisera). Samples were placed on microscopic slides in PBS:glycerine (1:1). As a positive control, samples were incubated with 3-morpholino-sydnonimine (SIN-1, 1 mM in TBSA–BSAT) for 1 h prior to the labeling process. Urate at 2 mM concentration (prepared in distilled water) was applied for 1 h prior to the labeling process in order to quench endogenous peroxynitrite. All microscopic analysis was accomplished under a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss) equipped with a digital camera (AxiocamHR, HQ CCD, Carl Zeiss). Filter set 10 (excitation, 450–490; emission, 515–565 nm) was used for FDA, DAF-FM, DHR and FITC, filter set 9 (excitation, 450–490 nm; emission 515–∞ nm) for DHE and filter set 49 (excitation, 365 nm; emission, 445/50 nm) was applied for UV autofluorescence. Pixel intensity was measured in area of circles using Axiovision Rel. 4.8 software (Carl Zeiss). The radii of circles were set to cover the largest sample area. Immunofluorescent detections were carried out on two separate plant generations with 5–6 plants examined in each (n = 5–6). Detection of nitrated proteins using SDS–PAGE and Western blot Protein extracts were prepared as described earlier. To evaluate the electrophoresis and transfer, we used Coomassie Brilliant Blue R-350 according to Welinder and Ekblad (2011). As a protein standard, actin from bovine liver (Sigma-Aldrich, cat. No. A3653) was used (Supplementary Fig. S8). Silver staining was carried out as previously described. A 25 µg aliquot of denaturated root and shoot protein was subjected to SDS–PAGE on 12% acrylamide gels. The proteins were transferred to PVDF membranes using the wet blotting procedure (25 mA, 16 h) for immunoblotting. After transfer, membranes were used for cross-reactivity assays with rabbit polyclonal antibody against 3-nitrotyrosine diluted 1:2,000. Immunodetection was performed by using affinity-isolated goat anti-rabbit IgG–alkaline phosphatase secondary antibody at a dilution of 1:10,000, and bands were visualized by using the NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate) reaction. Nitrated BSA served as the positive control. Western blot was applied to two separate protein extracts from different plant generations, multiple times per extract, giving a total of six blotted membranes (n = 2). Statistical analysis Root morphological data (Fig. 4) were analyzed using STATISTICA 10.0 software. To ascertain the effect of Se treatment on the anatomical parameters examined, one-way analysis of variance (ANOVA) was applied. Since most of the data showed non-normal distribution, we used a non-parametric test (Kruskal–Wallis ANOVA) to test the differences of means. In order to determine the relationship between Se concentration and the measured parameters, a non-parametric analysis of correlation (Spearman’s rank order correlation) was used. Data are given as mean values ± SD; the level of significance was *P < 0.05, **P < 0.01 and ***P < 0.001. In the case of any additional data, the results are shown as the mean ± SE. Data were statistically evaluated by Duncan’s multiple range test (one-way ANOVA, P ≤ 0.05) using SigmaPlot 12 or by Student’s t-test applying Microsoft Excel 2010. Funding This work was supported by the Hungarian Academy of Sciences [János Bolyai Research Scholarship grant No. BO/00751/16/8]; the National Research, Development and Innovation Fund [grant No. NKFI-6, K120383]; the EU-funded Hungarian grant [EFOP-3.6.1-16-2016–00008]; and the Ministry of Human Capacities [UNKP-17-4 New National Excellence Program to Z.K.]. Acknowledgments The authors thank Professor Aaron Chang (Kaohsiung Medical University, Graduate Institute of Natural Products, Kaohsiung, Taiwan) for the Astragalus membranaceus (Fisch.) Bunge seeds. We are also grateful to Dr. Attila Pécsváradi (Department of Plant Biology, University of Szeged) for his valuable advice and help. Disclosures The authors have no conflicts of interest to declare. References Arasimowicz-Jelonek M. , Floryszak-Wieczorek J. 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Google Scholar Crossref Search ADS PubMed Abbreviations Abbreviations BSA bovine serum albumin CAT catalase DAF-FM DA 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate DHE dihydroethidium DHR dihydrorhodamine 123 DPI diphenylene iodonium FDA fluorescein diacetate FITC fluorescein isothiocyanate GSNO S-nitrosoglutathione GSNOR S-nitrosoglutathione reductase H2O2 hydrogen peroxide NBT nitroblue tetrazolium NBT nitroblue tetrazolium NO nitric oxide NO2 nitrogen dioxide radical N2O3 dinitrogen-trioxide N2O4 dinitrogen tetroxide NOX NADPH oxidase NR nitrate reductase O2– superoxide OH hydroxyl radical ONOO– peroxynitrite PBS phosphate-buffered saline POD peroxidase RNS reactive nitrogen species ROS reactive oxygen species Se selenium SIN-1 3-morpholino-sydnonimine SMT selenocysteine methyltransferase SNO S-nitrosothiol SOD superoxide dismutase © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Nitro-Oxidative Stress Correlates with Se Tolerance of Astragalus Species

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy099
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Abstract

Abstract At high concentrations, selenium (Se) exerts phytotoxic effects in non-tolerant plant species partly due to the induction of nitro-oxidative stress; however, these processes are not fully understood. In order to obtain a more accurate view of the involvement of nitro-oxidative processes in plant Se sensitivity, this study aims to characterize and compare Se-triggered changes in reactive oxygen (ROS) and nitrogen species (RNS) metabolism and the consequent protein tyrosine nitration as a marker of nitrosative stress in the non-accumulator Astragalus membranaceus and the Se hyperaccumulator Astragalus bisulcatus. The observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth and cell viability) supported that A. membranaceus is Se sensitive while the hyperaccumulator A. bisulcatus tolerates high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism, leading to its accumulation. Furthermore, Se increased the production or disturbed the metabolism of RNS (nitric oxide, peroxynitrite and S-nitrosoglutathione), consequently resulting in intensified protein tyrosine nitration in sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se-induced ROS/RNS accumulation and tyrosine nitration proved to be negligible, suggesting that this species is able to prevent Se-induced nitro-oxidative stress. Introduction Selenium (Se) is a non-metal element which seems to be non-essential for higher plants. Still, its chemical similarity to sulfur (S) results in its uptake and metabolism via S transporters and pathways (Pilon-Smits and Quinn 2010). Moreover, a few plant species not only take up but also accumulate or hyperaccumulate high Se levels in their tissues. The ability for Se hyperaccumulation has been described in 45 plant taxa in six families (White 2016). The Astragalus (Fabaceae) genus is the most representative since a large number of species (25) in the genus have the ability to take up and tolerate high concentrations of Se (Shrift 1969). Species such as Astragalus bisulcatus grow on seleniferous soils and can accumulate >1,000 μg g−1 DW Se (up to 1% of its dry weight). Hyperaccumulators possess 10- to 100-fold higher endogenous Se content as well as a higher Se:S ratio compared with non-accumulators (White et al. 2007). Another distinctive feature of hyperaccumulators is the active sulfate/selenate assimilation which is suggested by the dominance of organic Se forms (γ-glutamyl-methyl-selenocysteine) in their tissues. Hyperaccumulators can be characterized by notable root to shoot Se translocation (Mehdawi and Pilon-Smits 2012). Species such as A. bisulcatus are able to sequester Se in their epidermis and trichomes, which may have a role both in defense and Se stress mitigation (Freeman et al. 2006). The mechanism responsible for Se hyperaccumulation is the constitutive expression of several SULTR transporters, which contributes to the preferential uptake of selenate over sulfate (Cabannes et al. 2011). Also, the expression of certain enzymes involved in selenate/sulfate assimilation is enhanced in hyperaccumulators, resulting in greater inorganic–organic conversion (Freeman et al. 2010). Moreover, hyperaccumulators express selenocysteine methyltransferase (SMT) which is responsible for the conversion of toxic selenocysteine to methyl-selenocysteine (Sors et al. 2009). Se tolerance is also typical for hyperaccumulators; however, the molecular mechanism of this ability is only partly understood. High tissue concentrations of inorganic Se forms can induce the production of reactive oxygen species (ROS) such as superoxide (O2·–), hydrogen peroxide (H2O2) and hydroxyl radical (OH·), leading to oxidative stress (Van Hoewyk 2013). The amount of the generated ROS and consequently the redox homeostasis is precisely controlled by antioxidant mechanisms. Beyond the enzymatic components such as superoxide dismutase (SOD), catalase (CAT) and peroxidases (PODs), non-enzymatic antioxidants such as ascorbate and glutathione (GSH) play crucial role in the defense against oxidative damage (Das and Roychoudhury 2014). For Se-induced ROS accumulation, GSH and its depletion seems to be responsible (Van Hoewyk 2013). According to previous data, hyperaccumulators prefer to produce organic Se forms presumably in order to avoid oxidative stress (Freeman et al. 2006, Van Hoewyk 2013). Besides ROS, reactive nitrogen species (RNS) are also formed as the effect of environmental stresses such as Se exposure (reviewed by Kolbert et al. 2016). This group of nitric oxide (NO)-related molecules consists of peroxynitrite (ONOO–), S-nitrosoglutathione (GSNO), dinitrogen trioxide (N2O3), dinitrogen tetroxide (N2O4) and nitrogen dioxide radical (·NO2) (Corpas et al. 2007). The overproduction of RNS leads to nitrosative stress during which one of the principle mechanisms is the nitration of tyrosine residues in certain proteins yielding 3-nitrotyrosine (Corpas et al. 2013). This modification causes structural and functional changes in the affected proteins. In most published cases, tyrosine nitration results in loss of activity of the target plant proteins (Kolbert et al. 2017) or it can negatively affect signal transduction through the prevention of tyrosine phosphorylation (Galetskiy et al. 2011). An Se-induced increase in protein tyrosine nitration and in oxidative parameters (ROS levels, lipid peroxidation and antioxidants) has been revealed in the leaves of non-accumulator pea (Lehotai et al. 2016). Also, the relationship between the toxicity of Se forms and protein tyrosine nitration has been evaluated in the non-accumulator Arabidopsis thaliana and the secondary accumulator Brassica juncea (Molnár et al. 2018a, Molnár et al. 2018b), but there is no knowledge about RNS metabolism and protein nitration in Se hyperaccumulator plants such as A. bisulcatus. Another species in the Astragalus genus is Astragalus membranaceus, which is considered to be pharmacologically relevant. The root of this Astragalus species has been used in Chinese medicine for thousands of years because of its general strengthening effect. Based on the literature, in modern medicine it can provide perspectives for the prevention and therapy of cerebrovascular, cardiovascular, neurodegenerative and liver diseases (Yang et al. 2013). Despite the significance of A. membranaceus, we know little about its Se accumulation and tolerance or about reactive species metabolism and nitrosative stress. Therefore, this comparative study aims to explore the possible differences in Se-modified ROS and RNS metabolism and the consequent protein tyrosine nitration using the hyperaccumulator A. bisulcatus and A. membranaceus as another species in the same genus. The better understanding of tolerance mechanisms of Se hyperaccumulator plant species is of particular significance in phytoremediation (Gupta and Gupta 2017) and in biofortification (Wu et al. 2015) as well from an ecological (Schiavon and Pilon-Smits 2017) point of view. Furthermore, examination of Se accumulation and tolerance of the medicinal herb A. membranaceus can have importance in aspects of human health. Results Selenium uptake, accumulation and microelement imbalance Selenate-induced Se accumulation showed differences in the organs of Astragalus species (Fig. 1). In the root tissues of A. membranaceus, the Se concentration was significantly enhanced as an effect of increasing exogenous selenate supplementation (Fig. 1A). In the case of A. membranaceus cotyledons, Se accumulation was not concentration dependent and proved to be lower compared with the root (Fig. 1B). The Se content measured in cotyledons of 50 or 100 µM selenate-treated A. membranaceus did not reach the endogenous Se content of the control A. bisulcatus. The root of the hyperaccumulator A. bisulcatus showed moderate Se accumulation (Fig. 1A), while in the cotyledons a remarkable, concentration-dependent increase of Se content was observed (Fig. 1B). In the case of 100 µM selenate supplementation, the accumulated Se exceeded 1,700 µg g–1 DW in the cotyledons of A. bisulcatus. It has to be mentioned that a significant difference was observed in the endogenous Se contents of untreated Astragalus plants. Cotyledons of A. bisulcatus contained 200-fold more Se than the same organs of A. membranaceus (Fig. 1B). Regarding the root, a similar but much smaller (16-fold) difference was revealed (Fig. 1A). Fig. 1 View largeDownload slide Concentration of selenium in the root system (A) and in the cotyledons (B) of 14-day-old A. membranaceus and A. bisulcatus treated with 0 (control), 50 or 100 µM sodium selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Fig. 1 View largeDownload slide Concentration of selenium in the root system (A) and in the cotyledons (B) of 14-day-old A. membranaceus and A. bisulcatus treated with 0 (control), 50 or 100 µM sodium selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Selenate exposure led to the modification of microelement concentrations in the organs of Astragalus species (Table 1). Of the examined microelements, the contents of the essential Fe, Zn, Mn and B showed a notable reduction, especially in the cotyledons of A. membranaceus. However, the concentration of the above-mentioned elements was not affected at all or only slightly changed by selenate in A. bisulcatus cotyledons. Regarding the root system, more serious effects were observed in the case of A. membranaceus compared with A. bisulcatus, e.g. the Fe concentration decreased by 30% in A. membranaceus but only by 15% in A. bisulcatus. In contrast to the other microelements, Mo concentrations in A. membranaceus organs significantly increased as an effect of Se treatments. In A. bisulcatus, the concentrations of Mo were decreased or were not modified by Se (Table 1). Table 1 Concentrations of Fe, Zn, Mn, B and Mo in the root system and cotyledons of 14-day-old Astragalus species treated with 0, 50 or 100 µM selenate for 14 d Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Different superscript letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Table 1 Concentrations of Fe, Zn, Mn, B and Mo in the root system and cotyledons of 14-day-old Astragalus species treated with 0, 50 or 100 µM selenate for 14 d Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Fe (µg g-1 DW) Zn (µg g-1 DW) Mn (µg g-1 DW) B (µg g-1 DW) Mo (µg g-1 DW) Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se Control 50 µM Se 100 µM Se A. membranaceus Cotyledon 133.20 ± 1.9a 65.35 ± 0.6b 59.81 ± 1.6c 101.50 ± 1.2a 72.18 ± 1.4b 65.54 ± 0.3c 106.61 ± 0.4a 85.94 ± 0.7b 68.22 ± 1.1c 113.84 ± 0.7a 53.22 ± 0.5b 39.61 ± 1.0c 5.87 ± 0.08b 6.80 ± 0.1a 6.88 ± 0.09a Root 503.80 ± 8.3a 338.81 ± 11.3b 358.90 ± 16.3b 152.10 ± 1.6b 145.51 ± 2.1c 166.93 ± 6.6a 129.63 ± 1.8a 76.41 ± 0.9b 60.57 ± 1.1c 51.62 ± 0.2a 51.55 ± 1.3a 44.27 ± 1.0b 2.88 ± 0.1b 5.13 ± 0.09a 5.02 ± 0.2a A. bisulcatus Cotyledon 108.23 ± 2.4a 105.80 ± 3.4a 95.47 ± 0.4b 73.97 ± 0.7a 74.98 ± 0.6a 73.58 ± 0.7a 102.88 ± 4.9a 107.86 ± 0.6a 99.92 ± 0.2a 46.26 ± 1.9a 47.77 ± 0.2a 43.53 ± 0.5b 1.63 ± 0.4a 2.13 ± 0.2a 1.94 ± 0.2a Root 963.60 ± 25.7b 1234.00 ± 14.7a 811.70 ± 5.0c 336.00 ± 5.2a 344.62 ± 5.0a 295.28 ± 0.4b 147.3 ± 2.3a 90.08 ± 5.6b 96.65 ± 1.8b 47.35 ± 4.7a 31.68 ± 2.1b 40.63 ± 1.5a 3.08 ± 0.02a 2.24 ± 0.09b 2.21 ± 0.1b Different superscript letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Growth and Se tolerance of Astragalus species Se tolerance of Astragalus species was evaluated by germination capacity, biomass production, root meristem viability and root elongation on selenate-supplemented medium. Both species showed approximately 85% germination under control conditions, and this good germination capability was retained by A. bisulcatus on 50 and 100 µM selenate-treated plates (Fig. 2A). In contrast, the presence of selenate significantly and concentration dependently reduced the germination percentage of A. membranaceus. In the case of 100 µM Se treatment, 55% of A. membranaceus seeds placed on the medium were germinated, while A. bisulcatus showed better (∼70%) germination performance. Fig. 2 View largeDownload slide (A) Germination percentage of Astragalus species on agar media supplemented with 0 (control), 50 or 100 µM sodium selenate. Shoot (B) and root (C) fresh weight of 14-day-old A. membranaceus and A. bisulcatus plants treated with 0 (control), 50 or 100 µM selenate. Different letters indicate significant differences according to Duncan’s test (n = 15, P ≤ 0.05). (D) Representative images showing 14-day-old A. membranaceus and A. bisulcatus plants grown on control or 50 or 100 µM selenate-containing agar media. Photographs show three representative individuals per treatment. Scale bars = 3 cm. Fig. 2 View largeDownload slide (A) Germination percentage of Astragalus species on agar media supplemented with 0 (control), 50 or 100 µM sodium selenate. Shoot (B) and root (C) fresh weight of 14-day-old A. membranaceus and A. bisulcatus plants treated with 0 (control), 50 or 100 µM selenate. Different letters indicate significant differences according to Duncan’s test (n = 15, P ≤ 0.05). (D) Representative images showing 14-day-old A. membranaceus and A. bisulcatus plants grown on control or 50 or 100 µM selenate-containing agar media. Photographs show three representative individuals per treatment. Scale bars = 3 cm. With regard to biomass production, 14-day-old, untreated individuals of the species possessed a similar shoot weight (Fig. 2B). However, the root fresh weight of control A. membranaceus was significantly smaller (Fig. 2C) and the phenotype of the root system notably differed from that of A. bisulcatus (Fig. 2D). Both concentrations of exogenous Se (50 and 100 µM) negatively affected shoot (40% and 46% reduction, respectively) and root growth (57% and 75% reduction, respectively) of A. membranaceus (Fig. 2B, C) and a brown discoloration was visible on the root surface of Se-treated plants (Fig. 2D). In contrast, A. bisulcatus showed significantly slighter growth inhibition, since the root biomass was affected only by the highest Se dose (30% reduction) and none of the treatments inhibited shoot growth (Fig. 2B–D). Se tolerance correlates with the capability of maintaining primary root elongation; therefore, the Se tolerance index can be calculated from primary root length data (Tamaoki et al. 2008). Compared with the 100% tolerance of the untreated plants (indicated by a dashed line in Fig. 3A), 50 or 100 µM selenate resulted in a 35% or 25% tolerance index of A. membranaceus, respectively (Fig. 3A). However, A. bisulcatus was able to maintain its root growth, and Se even slightly increased elongation, resulting in tolerance indexes around or above 100%. Furthermore, we examined the Se tolerance of the species by evaluating the viability of the root meristem cells using fluorescein diacetate (FDA) staining (Fig. 3B, C). As expected from the previous data, the meristem cells of A. membranaceus showed 50% or 85% viability loss as the effect of 50 or 100 µM selenate exposure, respectively. Even though root elongation of A. bisulcatus was not negatively affected by any of the applied Se doses (Fig. 3A), root meristem cells suffered 50% viability loss as the effect of the highest Se concentration (Fig. 3B, C). We acknowledge that the use of plant tissues with highly reduced viability might limit the reliability of the data. At the same time, the choice of the 14 d treatment period proved to be necessary for the appearance of the effect, as well as for the emergence of tolerance in this comparative Astragalus system (Supplementary Fig. S1). Fig. 3 View largeDownload slide (A) Selenium tolerance indexes (%) of Astragalus species treated with 50 or 100 µM selenate for 14 d. The 100% tolerance index of untreated plants is indicated by a dashed line. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Viability of primary root meristem cells in control and selenate-treated Astragalus species. Significant differences were determined by Student’s t-test and indicated by asterisks (n = 15, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.= non-significant). (C) Representative microscopic images indicating root tips of control (C) and selenate-treated Astragalus species stained with fluorescein diacetate. Scale bars = 500 µm. Fig. 3 View largeDownload slide (A) Selenium tolerance indexes (%) of Astragalus species treated with 50 or 100 µM selenate for 14 d. The 100% tolerance index of untreated plants is indicated by a dashed line. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Viability of primary root meristem cells in control and selenate-treated Astragalus species. Significant differences were determined by Student’s t-test and indicated by asterisks (n = 15, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.= non-significant). (C) Representative microscopic images indicating root tips of control (C) and selenate-treated Astragalus species stained with fluorescein diacetate. Scale bars = 500 µm. Se-induced tissue-level changes in the roots To evaluate the Se-induced tissue-level changes in the root structure of both Astragalus species, we measured the diameter of the root, the thickness of the cortex and the diameter of the vascular cylinder (stele). Both untreated and Se-treated A. membranaceus plants had thick roots, and Se application did not significantly affect root diameter (F = 1.25, P = 0.29). In the case of control and 50 µM Se-treated plants, A. membranaceus had nearly twice as thick roots as A. bisulcatus (Fig. 4A). When 100 µM Se was added to the media, the roots of A. bisulcatus exhibited remarkable thickening, the degree of was similar to that of A. membranaceus. This tendency was also confirmed by analysis of correlation (r = 0.82, P < 0.001). Similarly, the sensitive A. membranaceus had a significantly thicker root cortex than the Se hyperaccumulator A. bisulcatus in both control and 50 µM Se-treated plants, but it was almost the same in the roots of 100 µM Se-treated plants of both species (Fig. 4B). Increasing Se levels significantly enhanced the thickness of the cortex in the case of A. bisulcatus (F = 403.88, P < 0.001; r = 0.88, P < 0.001), while a remarkable increase was found only in the root cortex of 50 µM Se-treated A. membranaceus (F = 33.88; P < 0.001; r = 0.34, P < 0.001). There was a remarkable increase of stele diameter in A. membranaceus roots exposed to 50 µM Se, while it significantly decreased compared with control after 100 µM Se application. The size of the stele in the roots of A. bisulcatus was only affected by the highest Se stress (Fig. 4C). The stele of control and 50 µM Se-treated A. membranaceus roots was at least twice as thick as that of A. bisulcatus. Se stress-induced deposition of callose was investigated in aniline blue-stained root sections taken from the mature zone. Significantly higher fluorescence was found in Se-treated roots of A. membranaceus compared with the control, while it diminished after Se application in A. bisulcatus (Fig. 4D). Lignin and suberin deposition was visualized using Auramine O staining in the root sections. An intense fluorescence was found in the stele in control roots of both species due to the xylem vessels (Fig. 4E). In the Se-treated roots of A. membranaceus, a slight fluorescence appeared on the surface (exodermis) of the roots. This staining affected both the endodermis and the exodermis in the roots of Se-treated A. bisulcatus. Fig. 4 View largeDownload slide Root diameter (A), the thickness of the cortex (B) and the diameter of the stele in the roots (C) of control (Cont) and 50 or 100 µM selenate-treated (50 Se and 100 Se) Astragalus species after 14 d. The values of aniline blue (AB) fluorescence (pixel intensity) which refers to callose deposition are given in Control% (D). Auramine O staining of the control and Se-treated root sections of both species (E). Strong fluorescence can be seen at the xylem vessels (white arrows) and the endodermis and/or the exodermis (red arrows). Scale bar = 100 µm. Different letters refer to significant differences among the treatments within the same species according to Kruskal–Wallis ANOVA at P < 0.05 (n = 6). Significant differences between the species within the same treatment were determined by Mann–Whitney U-test and are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant). Fig. 4 View largeDownload slide Root diameter (A), the thickness of the cortex (B) and the diameter of the stele in the roots (C) of control (Cont) and 50 or 100 µM selenate-treated (50 Se and 100 Se) Astragalus species after 14 d. The values of aniline blue (AB) fluorescence (pixel intensity) which refers to callose deposition are given in Control% (D). Auramine O staining of the control and Se-treated root sections of both species (E). Strong fluorescence can be seen at the xylem vessels (white arrows) and the endodermis and/or the exodermis (red arrows). Scale bar = 100 µm. Different letters refer to significant differences among the treatments within the same species according to Kruskal–Wallis ANOVA at P < 0.05 (n = 6). Significant differences between the species within the same treatment were determined by Mann–Whitney U-test and are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ns = non-significant). Se-induced changes in ROS and RNS metabolism in root and shoot tissues One possible molecular mechanism of Se phytotoxicity is the production of ROS and the consequent oxidative stress (Van Hoewyk 2013). Recently, Se-triggered nitrosative processes have also been discovered, which are caused by the disturbance of RNS metabolism (Kolbert et al. 2016). The ROS- and RNS-inducing effects of Se were compared in the organs of Astragalus species (Fig. 5) in order to reveal the possible link between Se tolerance or sensitivity and Se-induced oxidative and nitrosative (together with nitro-oxidative) stress. Fig. 5 View largeDownload slide (A) The level of superoxide in the root tips of A. membranaceus and A. bisulcatus treated with 0, 50 or 100 µM selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Representative fluorescent microscopic images showing DHE-stained root tips of Astragalus species. Scale bars = 500 µm. (C) Representative photographs taken from NBT-stained cotyledons of control (0 Se), 50 or 100 µM selenate-treated A. membranaceus and A. bisulcatus. The blue discoloration refers to superoxide accumulation. Scale bar = 1 cm. (D) Native-PAGE (10%) separation of NOX isoenzymes in cotyledon and root of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. The most representative protein band is indicated as ‘main band’. Additional putative isoenzymes are indicated by black arrows, and newly appeared NOX isoenzymes are labeled by asterisks. (E and F) Total activity of SOD enzymes in the organs of Astragalus species supplemented (50 or 100 µM) or not (0 µM) with selenate. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). (G) Native-PAGE separation (10%) of SOD isoenzymes in cotyledon and root of control and selenate-treated Astragalus species. Fig. 5 View largeDownload slide (A) The level of superoxide in the root tips of A. membranaceus and A. bisulcatus treated with 0, 50 or 100 µM selenate for 14 d. Different letters indicate significant differences according to Duncan’s test (n = 10, P ≤ 0.05). (B) Representative fluorescent microscopic images showing DHE-stained root tips of Astragalus species. Scale bars = 500 µm. (C) Representative photographs taken from NBT-stained cotyledons of control (0 Se), 50 or 100 µM selenate-treated A. membranaceus and A. bisulcatus. The blue discoloration refers to superoxide accumulation. Scale bar = 1 cm. (D) Native-PAGE (10%) separation of NOX isoenzymes in cotyledon and root of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. The most representative protein band is indicated as ‘main band’. Additional putative isoenzymes are indicated by black arrows, and newly appeared NOX isoenzymes are labeled by asterisks. (E and F) Total activity of SOD enzymes in the organs of Astragalus species supplemented (50 or 100 µM) or not (0 µM) with selenate. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). (G) Native-PAGE separation (10%) of SOD isoenzymes in cotyledon and root of control and selenate-treated Astragalus species. In root tips of A. membranaceus, both Se concentrations increased superoxide levels, although the highest and significant superoxide production was observed in the case of 50 µM Se, resulting in a 170% increase (Fig. 5A, B). In the root tips of tolerant A. bisulcatus, selenate had no effect on superoxide levels (Fig. 5A, B). In intact cotyledons, superoxide levels were examined qualitatively by nitroblue tetrazolium (NBT) staining (Fig. 5C). In the case of 50 or 100 µM Se-treated A. membranaceus plants, the intense presence of blue colorization indicated superoxide production. In A. bisulcatus, slightly intensified blue staining was detected only as the effect of 100 µM Se treatment (Fig. 5C). In order to reveal the mechanism of the different superoxide responses of the species, we examined the metabolism of this reactive intermediate. The superoxide-generating NADPH oxidase (NOX) isoenzymes were separated by native-PAGE and a protein band which was strongly present in all samples was determined (Fig. 5D ‘main band’). In the cotyledons of A. bisulcatus one, while in A. membranaceus four additional putative NOX isoenzymes were detected. As the effect of Se, only slight changes occurred in NOX isoenzyme activities especially in A. bisulcatus, while more protein bands showed increased activity in A. membranaceus cotyledons (Fig. 5D;Supplementary Fig. S3). In the roots of both species, the activity of the main NOX protein band was less pronounced, although Se induced its activity in A. bisulcatus roots. In addition to the main protein band, four other isoenzymes were detected in A. bisulcatus roots, three of which showed induction as the effect of selenate exposure (Fig. 5D;Supplementary Fig. S3). In the case of A. membranaceus roots, Se reduced the activity of the main NOX band, which seemed to be substituted by the appearance and strong activation of additional, putative NOX isoenzymes (indicated by asterisks in Fig. 5D and Supplementary Fig. S3). Both concentrations of selenate caused notable (∼30% and 38%) induction of superoxide-eliminating SOD enzymes in A. membranaceus roots, while the effect of selenate in the root system of A. bisulcatus proved to be much slighter (∼10%, Fig. 5E). Regarding the cotyledons, selenate exposure resulted in SOD activation only in A. membranaceus, and the effect proved to be slighter compared with the root (∼15%, Fig. 5F). We separated SOD isoforms by native-PAGE, and differences were observed between the species and also between the organs (Fig. 5E). In both organs of A. bisulcatus, four activity bands (Mn SODI, Fe SOD I, Fe SOD II and Cu/Zn SOD) were identified, while in A. membranaceus cotyledons six bands were detected (Mn SODII, Fe SOD I, Fe SOD II, Cu/Zn SOD I, Cu/Zn SOD II and Cu/Zn SOD III). Moreover, in the root system of A. membranaceus, only three SOD activity bands (Mn SODII, Fe SODI and Fe SODII) were observed. Quantification showed that selenate at the higest applied concentration exerted a slight effect on SOD isoenzymes in A. bisulcatus cotyledons (Supplementary Fig. S4). In contrast, five SOD isoforms out of six showed intensified activity as the effect of 50 µM Se in cotyledons of A. membranaceus. Regarding the root system, both applied Se treatments induced the activity of Mn, Fe and Cu/Zn SODs in A. bisulcatus, but these inductions were much more intense in A. membranaceus (Supplementary Fig. S4). Similar to superoxide, NO formation was significantly enhanced as an effect of 50 µM Se in the root of sensitive A. membranaceus (Fig. 6A, B). Regarding peroxynitrite, 50 µM Se resulted in its accumulation, but the highest Se dose decreased its level in the root tips of the sensitive species (Fig. 6E, F). Interestingly, none of the applied Se treatments had any observable effect on the examined RNS levels in A. bisulcatus root tips (Fig. 6A, B, E, F). Fig. 6 View largeDownload slide The level of nitric oxide (A–D) and peroxynitrite (E–H) in intact root tips (A, B, E, F) and cotyledon cross-sections (C, D, G, H) of control (0 µM), 50 µM or 100 µM selenate-treated A. membranaceus and A. bisulcatus. Scale bars = 500 µm. (I–L) Immunofluorescent detection of GSNO in cross-sections of roots (I and J) and cotyledons (K and L). Scale bars = 200 µm. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). (M) Native-PAGE (6%) of Astragalus cotyledon and root extracts and staining for GSNOR activity. Astragalus membranaceus and A. bisulcatus were treated with 0, 50 or 100 µM selenate for 14 d. Fig. 6 View largeDownload slide The level of nitric oxide (A–D) and peroxynitrite (E–H) in intact root tips (A, B, E, F) and cotyledon cross-sections (C, D, G, H) of control (0 µM), 50 µM or 100 µM selenate-treated A. membranaceus and A. bisulcatus. Scale bars = 500 µm. (I–L) Immunofluorescent detection of GSNO in cross-sections of roots (I and J) and cotyledons (K and L). Scale bars = 200 µm. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). (M) Native-PAGE (6%) of Astragalus cotyledon and root extracts and staining for GSNOR activity. Astragalus membranaceus and A. bisulcatus were treated with 0, 50 or 100 µM selenate for 14 d. Unlike the roots, both species showed NO accumulation in their cotyledons as the effect of 50 µM Se (Fig. 6C, D). Both Se concentrations triggered significant peroxynitrite generation in the cotyledons of A. membranaceus, while in the tolerant species only slight, non-significant changes were observed (Fig. 6G, H). Se-induced alterations in GSNO levels were also determined in the root and shoot tissues of the species (Fig. 6I–L). Under control conditions, significantly higher GSNO content was determined in both organs of A. bisulcatus compared with A. membranaceus. Selenate treatments caused significant reduction in GSNO levels of both A. bisulcatus organs. A similar Se-induced diminution of GSNO content was found in A. membranaceus roots (Fig. 6I, J); however, in the cotyledons, Se exposure led to a significant and concentration-dependent increase of GSNO levels (Fig. 6K, L). Significantly increased fluorescence was detected in GSNO-pre-treated sections, which served as positive controls, while light-inactivated GSNO did not result in an increase in fluorescence (Supplementary Fig. S7). In their cotyledons, both species showed relatively high S-nitrosoglutathione reductase (GSNOR) activity compared with the root system during control conditions (Fig. 6M;Supplementary Fig. S6). Selenate exerted an inhibitory effect on GSNOR activity in A. bisulcatus cotyledons, while it notably induced it in the cotyledons of 50 µM selenate-treated A. membranaceus. As for the control root system, A. bisulcatus showed higher GSNOR activity than A. membranaceus where the activity was barely detectable (Fig. 6M;Supplementary Fig. S6). In the case of A. bisulcatus, selenate exerted a concentration-dependent reducing effect on GSNOR activity. In contrast to this, selenate did not modify the enzyme activity in the root of A. membranaceus (Fig. 6M;Supplementary Fig. S6). Selenium-induced protein tyrosine nitration Protein tyrosine nitration as a consequence of RNS accumulation was investigated by both immunofluorescence (Fig. 7) and Western blot analysis (Fig. 8). In cross-sections of A. membranaceus primary roots, an immunofluorescent signal related to 3-nitrotyrosine was observable mainly in the endodermal cell layer and within the central cylinder (Fig. 7B). Se exposure led to a significant increase in the 3-nitrotyrosine-dependent fluorescent signal in all tissues of the root (Fig. 7A), but this elevation was the most pronounced in the central cylinder (Fig. 7B). Under control conditions, 3-nitrotyrosine was located mainly in the endodermal cell layer of A. bisulcatus roots (Fig. 7B). Milder Se treatment caused a slight increase of the fluorescence in the endodermis, and the most serious Se exposure induced 3-nitrotyrosine accumulation in all tissues of the primary root, although this increase was smaller than in A. membranaceus roots (Fig. 7A). In cotyledons, Astragalus species showed differences in physiological 3-nitrotyrosine levels, since A. bisulcatus showed higher 3-nitrotyrosine-related fluorescence (Fig. 7C). Moreover, high levels of 3-nitrotyrosine were found to be located in cotyledon veins (Fig. 7D). Both selenate treatments significantly decreased the 3-nitrotyrosine content of A. bisulcatus cotyledons, but in the case of A. membranaceus, 100 µM selenate induced 3-nitrotyrosine formation (Fig. 7C). As positive and negative controls, sections were treated with SIN-1, and enhanced fluorescence intensity was detected, while urate pre-treatment remarkably mitigated 3-nitrotyrosine-dependent fluorescence (Supplementary Fig. S7). Fig. 7 View largeDownload slide The intensity of 3-nitrotyrosine-related fluorescence in root (A) or cotyledon (C) cross-sections of control and selenate-treated A. membranaceus and A. bisulcatus. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). Representative fluorescent microscopic images showing cross-sections of roots (B) and cotyledons (D) of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. Scale bars = 200 or 500 µm. Fig. 7 View largeDownload slide The intensity of 3-nitrotyrosine-related fluorescence in root (A) or cotyledon (C) cross-sections of control and selenate-treated A. membranaceus and A. bisulcatus. Different letters indicate significant differences according to Duncan’s test (n = 5–6, P ≤ 0.05). Representative fluorescent microscopic images showing cross-sections of roots (B) and cotyledons (D) of Astragalus species treated with 0, 50 or 100 µM selenate for 14 d. Scale bars = 200 or 500 µm. Fig. 8 View largeDownload slide Protein and tyrosine nitration pattern in cotyledon and root of control and selenate-treated Astragalus species (25 µg per lane). Silver-stained SDS gels (12%) and Western blots probed with a rabbit anti-nitrotyrosine polyclonal antibody (1:2,000). Commercial nitrated BSA (NO2-BSA) was used as a positive control, and the molecular marker is shown as a protein weight indicator. Gray arrows indicate intensification of nitration, and white arrows show protein bands with decreased nitration. Selenate-induced, newly appeared protein bands are indicated by black arrows. Fig. 8 View largeDownload slide Protein and tyrosine nitration pattern in cotyledon and root of control and selenate-treated Astragalus species (25 µg per lane). Silver-stained SDS gels (12%) and Western blots probed with a rabbit anti-nitrotyrosine polyclonal antibody (1:2,000). Commercial nitrated BSA (NO2-BSA) was used as a positive control, and the molecular marker is shown as a protein weight indicator. Gray arrows indicate intensification of nitration, and white arrows show protein bands with decreased nitration. Selenate-induced, newly appeared protein bands are indicated by black arrows. In the whole protein extract, tyrosine nitration was determined by Western blot analysis (Fig. 8). In A. membranaceus cotyledons, selenate intensified tyrosine nitration of five protein bands (∼27, 22, 17, 12 and 10 kDa, indicated by gray arrows) but a newly nitrated protein band could not be observed. In cotyledons of A. bisulcatus, both Se treatments resulted in the appearance of a highly nitrated protein band (with high molecular weight, indicated by black arrows) but Se did not cause any other nitration-related change in the proteome. In A. bisulcatus roots, Se did not intensify protein tyrosine nitration, and even caused a decrease in three protein bands (∼75, 12 and 10 kDa). In contrast, the Se-sensitive Astragalus species showed several protein bands whose immunopositivity towards anti-3-nitrotyrosine showed an Se-dependent appearance. Discussion Both species were able to take up selenate from the external media (Fig. 1). Even though A. membranaceus accumulated a large amount of Se in its root, the root to shoot Se translocation proved to be slight. In contrast, in A. bisulcatus cotyledons, >7-fold Se concentrations were measured compared with A. membranaceus, indicating a high rate of Se translocation. Indeed, the root to shoot Se ratio was 3.8 in A. bisulcatus plants grown on 100 µM selenate, suggesting that it is a hyperacumulator species (Freeman et al. 2010). Furthermore, the relatively high endogenous Se content in the organs of control A. bisulcatus indicates its hyperaccumulator nature. Also the amount of the accumulated Se (∼1,800 µg g–1 DW in the cotyledons of 100 µM selenate-exposed plants) supports the hyperaccumulation capability of A. bisulcatus (Mehdawi and Pilon-Smits 2012). In addition to Se, exogenous selenate affected the concentrations of essential microelements such as Fe, Zn, Mn and B (Table 1) especially in A. membranaceus, inhibiting their absorption and consequently causing disturbances in their homeostasis. Similar antagonism between Se and macro- or microelements has earlier been described by others (Pazurkiewicz-Kocot et al. 2003, Filek et al. 2010, Zembala et al. 2010). Reduced availability of essential microelements may worsen the growth and physical condition of the plant. B is needed to maintain cell wall integrity, while Zn protects membrane lipids and proteins, and, together with Mn, Cu and Fe, is the metal component of SOD antioxidant enzymes (Cakmak 2000). In the case of the Se hyperaccumulator A. bisulcatus, the microelement homeostasis seems to be more stable, since Se did not cause disturbance in it, which may contribute to the better tolerance of this species. Se negatively affected the germination capability and the biomass production of young A. membranaceus, but the germination and growth of A. bisulcatus proved to be insensitive to Se (Fig. 2). However, root elongation concentration dependently decreased as the effect of elevated Se concentrations, suggesting the higher sensitivity of the root system to Se compared with the aerial plant parts (Lehotai et al. 2016). Because of the Se concentration-dependent response of elongation, root growth can be used as an indicator of Se tolerance (Molnár et al. 2018a, Tamaoki et al. 2008). The hyperaccumulator A. bisulcatus was able to maintain its root growth on Se-containing medium (Fig. 3A) even though meristem cells suffered a certain degree of loss of viability (Fig. 3B, C). The reduced root elongation (Fig. 3A) and meristem viability (Fig. 3B, C) of A. membranaceus indicate its sensitivity to Se. Beyond the viability of the root apical meristem, in the background of Se-inhibited organ development, the disturbances of hormone homeostasis or unfavorable alterations in primary metabolism can also be determined (reviewed by Kolbert et al. 2016). Based on the observed parameters (germination, biomass production, root elongation and cell viability), young A. membranaceus proved to be Se sensitive, while the hyperaccumulator A. bisulcatus showed remarkable Se tolerance, which supports the previously described connection between Se hyperaccumulation and (hyper)tolerance (Mehdawi and Pilon-Smits 2012). The main reason for Se tolerance of A. bisulcatus is that this species expresses the SMT enzyme which prevent toxic seleno-amino acid formation (Neuhierl and Bock 1996). Considering the high shoot Se accumulation (Fig. 1B), it can be assumed that the notable Se tolerance of A. bisulcatus is due to detoxification and not exclusion. We observed Se-induced alterations in root structure of both Astragalus species. Thicker roots of control and 50 µM Se-treated sensitive A. membranaceus compared with A. bisulcatus were probably due to the thicker cortex (Fig. 4A, B). The increment of the root diameter, including the thickening of the cortex is common in heavy metal-stressed plant roots (Arduini et al. 1995, Maksimović et al. 2007, Potters et al. 2007). The hyperaccumulator species A. bisulcatus showed more intense Se-induced root thickening than A. membranaceus (Fig. 4A–C), which is in agreement with the results of Li et al. (2009) where in the hyperaccumulating ecotype of Sedum alfredii, a lead/zinc-triggered increment in root diameter and other root morphological parameters was observed. The deposition of callose seems to be a good marker of stress-induced cell wall alterations. It was formerly found that copper can induce callose formation in onion epidermal cells and in the root tips of Brassica species (Kartusch 2003, Feigl et al. 2013). In our study, the sensitive Astragalus species showed both Se-triggered callose accumulation (Fig. 4D) and exodermal suberin lamellae deposition (Fig. 4E; Dalla Vecchia et al. 1999, Rahoui et al. 2017) which together may serve as an extracellular barrier limiting water and mineral uptake. This may result in Se exclusion and at the same time the inhibition of growth. In the case of A. bisulcatus, not only the exodermis but also the endodermis exhibited the presence of suberin (Fig. 4E). Since exodermal suberin deposition occurs earlier in time followed by the appearance of endodermal suberin as the effect of metal stress (Vaculík et al. 2012), we can conclude that in the case of A. membranaceus, the delayed formation of Se-induced endodermal suberin lamellae is associated with Se sensitivity. Moreover, the development of apoplastic barriers (exodermal and endodermal) can be considered as an adaptive trait (Vaculík et al. 2012). For the toxic effect of Se, the accumulation of ROS and the consequent oxidative stress is partly responsible (Van Hoewyk 2013). The accumulation of the rapidly generating, harmful ROS, superoxide anion (Fig. 5A–C), as well as the induction of SOD activity (Fig. 5E, F) suggest Se-triggered oxidative stress in A. membranaceus organs, while no sign of serious oxidative damage was observed in A. bisulcatus. The expression of superoxide-generating NOX isoenzymes showed species specificity in A. membranaceus roots, and newly expressed NOX isoenzymes were observed as the effect of selenate (Fig. 5D). Regarding SOD isoenzymes, A. membranaceus cotyledons express more Cu/Zn SODs than A. bisulcatus, and selenate remarkably increased the activity of most of the isoenzymes (Fig. 5G). Se-triggered superoxide accumulation has been observed in the non-accumulatord Stanleya albescens and Arabidopsis thaliana and in secondary accumulators such as Brassica napus, Brassica rapa and Brassica juncea (Molnár et al. 2018a, Molnár et al. 2018b, Tamaoki et al. 2008, Freeman et al. 2010, Chen et al. 2014, Dimkovikj and Van Hoewyk 2014). In the hyperaccumulator species Stanleya pinnata, elevated levels of ROS-scavenging compounds (ascorbate and GSH) were observed which are involved in the prevention of Se-induced oxidative stress (Freeman et al. 2010). In our study, A. bisulcatus showed moderately higher SOD activities (especially Cu/Zn SODs) in the roots compared with A. membranaceus (Fig. 5F) which may contribute to endurance against Se-induced oxidative stress. At the same time, Se hyperaccumulators are known to accumulate organic Se forms (mainly methyl-seleno-cysteine) instead of the oxidative stress-inducing inorganic Se compounds, which may be a relevant protection mechanism against oxidative stress (Schiavon and Pilon-Smits 2017). Additionally, Se exposure has been shown earlier to disturb the metabolism of RNS. A milder selenate dose triggered NO production mainly in the non-accumulator species (Fig. 6A–D) similarly to selenite-exposed Pisum sativum (Lehotai et al. 2016) or the selenate-treated secondary accumulator B. rapa (Chen et al. 2014). Based on the results of Rios et al. (2010), it is conceivable that selenate induces nitrate reductase (NR) which is the main enzymatic NO source in the root system and is also involved in NO production in the aerial plant parts (Zhang et al. 2011). The effect of Se on NR activity can be direct or indirect since Se-induced S deficiency may increase Mo content, thus inducing NR (Shinmachi et al. 2010, Yu et al. 2010). In our experiments, significantly higher Mo concentrations were measured in both organs of selenate-treated A. membranaceus (Table 1) which can be connected to the elevated NO production. Peroxynitrite can be formed in vivo in the fast reaction between superoxide radical and NO (Kissner et al. 1997), thus their accumulation may predict and explain Se-induced ONOO– generation. The concentration of this strong oxidative and nitrosative agent could reflect overall stress severity (Arasimowicz-Jelonek and Floryszak-Wieczorek 2011); therefore, we can suspect that A. membranaceus suffers more severe Se-triggered nitro-oxidative stress compared with A. bisulcatus. However, like the effect of the highest Se dose in A. membranaceus root, the peroxynitrite level decreases (Fig. 6E) due to the possible activation of scavenging mechanisms. GSNO is a mobile form of NO storage in plants, being responsible for protein S-nitrosylation. The spontaneous decomposition of GSNO leads to NO production, while it is enzymatically reduced by GSNOR or it can catalyze the transnitrosylation of protein thiols leading to its decomposition (Begara-Morales et al. 2018, Lindermayr 2018). Both species responded to the presence of selenate by decreasing the endogenous GSNO reservoir of their roots (Fig. 6I); however, this resulted in NO accumulation only in A. membranaceus (Fig. 6A). Presumably, in A. bisulcatus the originally high GSNO content participated in transnitrosylation reactions with cysteine thiols in proteins leading to S-nitrosothiol (SNO) formation, and GSNOR-catalyzed reduction is not involved in GSNO metabolism under Se stress. In the cotyledon of A. bisulcatus, the level of GSNO decreased (Fig. 6K, L) possibly due to spontaneous decomposition yielding NO but not GSNOR activity. Similarly to other species (reviewed by Corpas et al. 2013), both A. membranaceus and A. bisulcatus can be characterized by a certain physiological nitropoteome which means that a part of their protein pool is nitrated even in the control state. Both the Se-induced increase in fluorescence intensity (Fig. 7), and the presence of several newly nitrated protein bands (Fig. 8) indicated more intense protein tyrosine nitration in the organs of A. membranaceus compared with the hyperaccumulator A. bisulcatus. Moreover, both immunofluorescence and Western blot results showed that the tolerant species possesses a large physiological nitroproteome as well as large mobile NO storage form (GSNO) with which it is able to buffer NO radical content. The Se-induced GSNO and 3-nitrotyrosine decompositions without the accumulation of the reactive ·NO may contribute to tolerance against nitro-oxidative stress in A. bisulcatus. The Se-triggered decrease in the amount of 3-nitrotyrosine may be conceivable via proteasomal degradation (Castillo et al. 2015). Our experiments examined the sensitivity of young non-accumulator A. membranaceus and hyperaccumulator A. bisulcatus to Se in connection to secondary oxidative and nitrosative processes, and the obtained results are summarized in Fig. 9. As expected, the observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth and cell viability) indicated that A. membranaceus is Se sensitive while A. bisulcatus tolerates the presence of high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism involving NOXs and SODs, leading to superoxide accumulation. Furthermore, this study points out for the first time that Se induced the production or disturbed the metabolism of RNS (NO, ONOO– and GSNO) consequently resulting in intensified protein tyrosine nitration in the sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se decreased the high GSNO content and tyrosine nitroproteome without the accumulation of the NO radical, resulting in the lack of tyrosine nitration. These findings suggest that this species is able to prevent Se-induced nitro-oxidative stress to which enhanced ROS/RNS-scavenging capability may also contribute. Given that the elevated levels of other elements (e.g. zinc, arsenic and cadmium) have been reported to induce protein nitration and cause similar disturbances in ROS and RNS metabolism to Se (Leterrier et al. 2012, Feigl et al. 2015, Feigl et al. 2016, Liu et al. 2018), excess Se-induced nitro-oxidative stress can be considered a general rather than an Se-specific phenomenon. Future research should focus on the evaluation of the antioxidative system in order to obtain a more accurate view of nitro-oxidative processes in relation to Se tolerance. Fig. 9 View largeDownload slide Schematic model summarizing the data obtained in this study. In the sensitive species, selenium exposure induces intense modification of root cell wall structure, disturbs microelement homeostasis and induces NO, superoxide and peroxynitrite accumulation as well as protein tyrosine nitration (nitro-oxidative stress). The observed alterations together lead to selenium-induced damage. In contrast, the Se-tolerant species shows slight cell wall modifications and non-disturbed microelement homeostasis. Additionally, selenium does not trigger NO, superoxide, peroxynitrite or 3-nitrotyrosine formation; instead the high amount of endogenous NO storage (GSNO) and the large nitroproteome decrease without the accumulation of NO, suggesting that GSNO and/or the nitrosoproteome are able to buffer the amount of NO radical. In the hyperaccumulator, slight Se-triggered damage or the complete lack of damage can be observed. See details in the text. Abbreviations: 3NT = 3-nitrotyrosine. Fig. 9 View largeDownload slide Schematic model summarizing the data obtained in this study. In the sensitive species, selenium exposure induces intense modification of root cell wall structure, disturbs microelement homeostasis and induces NO, superoxide and peroxynitrite accumulation as well as protein tyrosine nitration (nitro-oxidative stress). The observed alterations together lead to selenium-induced damage. In contrast, the Se-tolerant species shows slight cell wall modifications and non-disturbed microelement homeostasis. Additionally, selenium does not trigger NO, superoxide, peroxynitrite or 3-nitrotyrosine formation; instead the high amount of endogenous NO storage (GSNO) and the large nitroproteome decrease without the accumulation of NO, suggesting that GSNO and/or the nitrosoproteome are able to buffer the amount of NO radical. In the hyperaccumulator, slight Se-triggered damage or the complete lack of damage can be observed. See details in the text. Abbreviations: 3NT = 3-nitrotyrosine. Materials and Methods Plant material and growing conditions Astragalus bisulcatus (Hook.) A. Gray seeds were obtained from B&T World Seeds (Aigues-Vives, France), and Astragalus membranaceus (Fisch.) Bunge seeds were provided by Professor Aaron Chang (Kaohsiung Medical University, Graduate Institute of Natural Products, Kaohsiung, Taiwan). Seeds were surface sterilized with 20% (v/v) sodium hypochlorite for 20 min, and washed with sterile distilled water four times in 20 min. Seeds were dried on a sterile metal filter and we polished them one by one using P-400 sanding paper in order to scratch the external seed coat. Seeds were placed on agar medium (the scratched surface of the seeds contacted the medium). Plastic, square Petri dishes contained half-strength Murashige and Skoog medium [0.8% (v/v) agar, 1% sucrose] supplemented with 0 (control), 50 or 100 μM sodium selenate (Na2SeO4). Both plant species were grown under controlled conditions (150 µmol m– s–1 photon flux density, 12 h/12 h light/dark cycle, relative humidity 55–60% and temperature 25 ± 2°C) for 14 d. All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Se and microelement content analysis Cotyledon and root materials of both Astragalus species were harvested separately and rinsed with distilled water then dried at 70°C for 72 h. Nitric acid [65% (w/v), Reanal] and H2O2 [30% (w/v) VWR Chemicals] were added to dried plant material. The samples were destroyed in a microwave destructor (MarsX-press CEM) at 200°C and 1,600 W for 15 min. After appropriate dilutions with distilled water, the samples were transferred to 20 ml Packard glasses. Element concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700 Series). Concentrations of Se and essential microelements (Fe, Zn, Mn, Mo are B) are given in µg g–1 DW. These analyses were carried out twice with three samples each (n = 3). Evaluation of germination, growth parameters, root cell viability and Se tolerance index Germinated seeds were counted in each Petri dish and germination percentages (%) were calculated. Fresh weights of root and shoot materials were measured using a balance, and the values are given in milligrams. Lengths of primary roots were measured manually. From the data, the Se tolerance index (%) was calculated according to the following formula: tolerance index (%) = (treated root length/mean control root length)×100 Cell viability in the root apical meristem was determined by using the FDA fluorophore. Root tips were incubated in 10 µM FDA solution (prepared in 10/50 mM MES/KCl buffer, pH 6.15) for 30 min in darkness and were washed four times in buffer. These data were acquired from three separate generations, and in each generation 15 plants/seeds were examined (n = 15). Evaluation of tissue-level changes in the roots induced by selenium Small pieces of root samples derived from the mature zone were fixed in 4% (w/v) paraformaldehyde according to Barroso et al. (2006). After the fixation, root samples were washed in distilled water and embedded in 5% agar (bacterial; Zelko et al. 2012 with modifications). Then 100 µm thick cross-sections were prepared using a vibratome (VT 1000S, Leica). The sections were placed on a slide with a drop of water and were stained with aniline blue [0.5% (w/v)] to detect the deposition of callose. The root sections were observed by a light microscope and an inverted fluorescent microscope (Zeiss Axiovert 200 M, Carl Zeiss) equipped with a digital camera (AxiocamHR, HQ CCD, Carl Zeiss). Images obtained by light microscopy were applied to measure several parameters of the root such as root diameter, the thickness of the cortex and the diameter of the stele according to Arduini et al. (1995). All data are given in micrometers. Fluorescent microscopy was applied to observe the fluorescence of secondary cell wall compounds such as lignin and suberin (Auramine O staining) as well as the formation of callose as a result of Se stress, using filter set 9 (excitation, 450–490 nm; emission, 515–∞ nm) and filter set 49 (excitation, 365 nm; emission 445/50 nm) (Feigl et al. 2013, Rahoui et al. 2017). In both cases, fluorescence intensity (pixel intensity) was measured on digital images applying Axiovision Rel. 4.8 software (Carl Zeiss) within circles of 100 µm radius which were set to cover the largest area of the vascular cylinder. The data of the Se-treated plants were calculated as a percentage of the control. These experiments were carried out on two separate plant generations with six plants examined in each (n = 6). In situ detection of ROS and RNS in the root tips and in cotyledons Dihydroethidium (DHE) at 10 µM concentration was applied for the detection of superoxide anion levels in the roots. Root segments were incubated for 30 min in darkness at 37°C, and washed twice with Tris–HCl buffer (10 mM, pH 7.4) (Kolbert et al. 2012). In cotyledons, instead of DHE, NBT was used for visualizing superoxide production. Excised cotyledons were incubated in Falcon tubes containing 5 ml of NBT solution (1 mg ml–1 in 10 mM phosphate buffer, pH 7.4) for 30 min under illumination. Pigments were removed by incubating the cotyledons in 80% (v/v) ethanol at 70°C for 30 min. The NO level of the root tips and in handmade cross-sections from cotyledons was monitored with the help of 4-amino-5-methylamino- 2′,7′-difluorofluorescein diacetate (DAF-FM DA) according to Kolbert et al. (2012). Root and cotyledon segments were incubated in 10 µM dye solution for 30 min (darkness, 25 ± 2°C), and washed twice with Tris–HCl (10 mM, pH 7.4). Peroxynitrite was also visualized in root tips and in handmade cross-sections of cotyledons. Samples were incubated in 10 µM dihydrorhodamine 123 (DHR) prepared in Tris–HCl buffer. After 30 min of incubation at room temperature, root tips and cotyledon segments were washed twice with the buffer solution (Sarkar et al. 2014). These analyses were carried out twice with 10 samples each (n = 10). Determining SOD, NADPH oxidase izoenzymes and GSNOR activity by native-PAGE Fresh cotyledon and root tissues of A. bisulcatus and A. membranaceus were ground with a double volume of extraction buffer (50 mM Tris–HCl buffer pH 7.6–7.8) containing 0.1 mM EDTA, 0.1% Triton X-100 and 10% glycerol, and centrifuged at 12,000 r.p.m. for 20 min at 4°C. The protein extract was treated with 1% protease inhibitor cocktail and stored at –20°C. Protein concentration was determined using the Bradford (1976) assay with bovine serum albumin (BSA) as a standard. In order to avoid the effect of the changes in protein concentration and composition induced by the treatments, our data are standardized to fresh weight by loading equal volumes of protein extracts in each well. Silver staining was performed according to Blum et al. (1987) with slight modifications. The gel was fixed with methanol and acetic acid, then treated with a sensitizing solution and staining solution containing AgNO3. The gel was developed in a solution containing sodium carbonate and formaldehyde (Supplementary Figs. S2, S5). NOX activity was examined on 10% native polyacrylamide gels by the NBT reduction method of López-Huertas et al. (1999) with slight modifications. In the case of cotyledons 15 µl and in the case of roots 25 µl of protein extracts were loaded in each well. Following electrophoresis, the gel was incubated in reaction buffer (50 mM Tris–HCl pH 7.4, 0.1 mM MgCl2, 1 mM CaCl2) containing 0.2 mM NBT and 0.2 mM NADPH for 20 min in darkness. As a positive control, the NOX-specific inhibitor diphenylene iodonium (DPI) was used at a final concentration of 50 µM. In addition, NADPH-independent superoxide production was examined on a gel without NAPDH supplementation. SOD activity was measured based on the ability of the enzyme to inhibit photochemical reduction of NBT catalyzed by riboflavin, as described by Dhindsa et al. (1981). A 250 mg aliquot of plant biomass was ground with 10 mg of polyvinyl polypyrrolidone (PVPP) in 1 ml of 50 mM pH 7.0 phosphate buffer containing 1 mM EDTA. The enzyme activity is expressed as specific activity (U g–1 FW weight), where 1 unit of SOD activity means 50% inhibition of NBT reduction in light. For the examination of SOD activity and isoenzymes, protein extracts (15 and 25 µl in the case of cotyledons and roots, respectively) were subjected to native gel electrophoresis on a 10% polyacrylamide gel (Beauchamp and Fridovich 1971). The gel was rinsed in 50 mM potassium phosphate buffer (pH 7.8) twice, ncubated for 20 min in 2.45 mM NBT in darkness and then for 15 min in freshly prepared 28 mM TEMED solution containing 2.92 µM riboflavin. After the incubation, the gels were washed twice and developed by light exposure. SOD isoforms were identified by incubating gels in 50 mM potassium phosphate containing 2 mM potassium cyanide to inhibit Cu/Zn SOD activity, or 5 mM H2O2 which inhibits Cu/Zn and Fe SOD activity for 30 min before staining with NBT. Mn SODs are resistant to both inhibitors. GSNOR activity was visualized using a slightly modified method of that by Seymour and Lazarus (1989). Native-PAGE was performed using 6% acrylamide gels with Tris-boric-EDTA buffer (8.9 mM Tris base, 8.9 mM boric acid and 0.2 mM Na2EDTA, pH 8). In the case of cotyledons 30 µl, and in case of roots 50 µl of protein extracts were loaded in each well. Gels were incubated for 15 min at 4°C in the presence of 2 mM NADH solution prepared in 100 mM sodium phosphate buffer (pH 7.4). Excess buffer was removed and a filter paper containing freshly prepared 3 mM GSNO solution (prepared in 100 mM sodium phosphate buffer, pH 7.4) was added (15 min, darkness, 4°C). NADH UV fluorescence was visualized at 312 nm wavelength using a gel documentation system (Image System Felix 1000/2000, Biostep). GSNOR enzyme activity consumed NADH, resulting in dark bands in the gel. Relevant bands showing NOX, SOD or GSNOR signals were quantified by Gelquant software (provided by biochemlabsolutions.com) and the data are presented as Supplementary Figs. S3, S4 and S6, respectively. These experiments were carried out on two separate plant generations with three samples examined each (n = 3). Immunofluorescent detection of GSNO and 3-nitrotyrosine in root and cotyledon cross-sections Cross-sections were prepared using a vibratome as described earlier, and immunodetection was performed according to Corpas et al. (2008) with slight modifications. Free-floating sections were incubated at room temperature overnight with rat antibody against GSNO (VWR Chemicals) diluted 1:2,500 in TBSA–BSAT solution containing 5 mM Tris buffer (pH 7.2), 0.9% (w/v) NaCl, 0.05% (w/v) sodium azide, 0.1% (w/v) BSA and 0.1% (v/v) Triton X-100. Samples were washed three times with TBSA–BSAT solution within 15 min. After the washings, cross-sections were incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-rat IgG secondary antibody (1:1,000 in TBSA–BSAT, Agrisera) for 1 h at room temperature. Samples were placed on microscopic slides in phosphate-buffered saline (PBS):glycerine (1:1). As a positive control, cross-sections were treated with 250 μM GSNO (prepared in TBSA–BSAT) for 1 h prior to the labeling process. Light-inactivated GSNO was prepared as described by Wodala and Horváth (2008) and was applied for 1 h prior to labeling. Immunodetection of 3-nitrotyrosine was carried out according to Valderrama et al. (2007). Samples were incubated for 3 d at 4°C with polyclonal rabbit antibody against 3-nitrotyrosine (Sigma-Aldrich) diluted in TBSA–BSAT (1:300). After three washes with TBSA–BSAT, sections were incubated for 1 h at room temperature in FITC-conjugated goat anti-rabbit IgG (1:1,000 in TBSA––BSAT, Agrisera). Samples were placed on microscopic slides in PBS:glycerine (1:1). As a positive control, samples were incubated with 3-morpholino-sydnonimine (SIN-1, 1 mM in TBSA–BSAT) for 1 h prior to the labeling process. Urate at 2 mM concentration (prepared in distilled water) was applied for 1 h prior to the labeling process in order to quench endogenous peroxynitrite. All microscopic analysis was accomplished under a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss) equipped with a digital camera (AxiocamHR, HQ CCD, Carl Zeiss). Filter set 10 (excitation, 450–490; emission, 515–565 nm) was used for FDA, DAF-FM, DHR and FITC, filter set 9 (excitation, 450–490 nm; emission 515–∞ nm) for DHE and filter set 49 (excitation, 365 nm; emission, 445/50 nm) was applied for UV autofluorescence. Pixel intensity was measured in area of circles using Axiovision Rel. 4.8 software (Carl Zeiss). The radii of circles were set to cover the largest sample area. Immunofluorescent detections were carried out on two separate plant generations with 5–6 plants examined in each (n = 5–6). Detection of nitrated proteins using SDS–PAGE and Western blot Protein extracts were prepared as described earlier. To evaluate the electrophoresis and transfer, we used Coomassie Brilliant Blue R-350 according to Welinder and Ekblad (2011). As a protein standard, actin from bovine liver (Sigma-Aldrich, cat. No. A3653) was used (Supplementary Fig. S8). Silver staining was carried out as previously described. A 25 µg aliquot of denaturated root and shoot protein was subjected to SDS–PAGE on 12% acrylamide gels. The proteins were transferred to PVDF membranes using the wet blotting procedure (25 mA, 16 h) for immunoblotting. After transfer, membranes were used for cross-reactivity assays with rabbit polyclonal antibody against 3-nitrotyrosine diluted 1:2,000. Immunodetection was performed by using affinity-isolated goat anti-rabbit IgG–alkaline phosphatase secondary antibody at a dilution of 1:10,000, and bands were visualized by using the NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate) reaction. Nitrated BSA served as the positive control. Western blot was applied to two separate protein extracts from different plant generations, multiple times per extract, giving a total of six blotted membranes (n = 2). Statistical analysis Root morphological data (Fig. 4) were analyzed using STATISTICA 10.0 software. To ascertain the effect of Se treatment on the anatomical parameters examined, one-way analysis of variance (ANOVA) was applied. Since most of the data showed non-normal distribution, we used a non-parametric test (Kruskal–Wallis ANOVA) to test the differences of means. In order to determine the relationship between Se concentration and the measured parameters, a non-parametric analysis of correlation (Spearman’s rank order correlation) was used. Data are given as mean values ± SD; the level of significance was *P < 0.05, **P < 0.01 and ***P < 0.001. In the case of any additional data, the results are shown as the mean ± SE. Data were statistically evaluated by Duncan’s multiple range test (one-way ANOVA, P ≤ 0.05) using SigmaPlot 12 or by Student’s t-test applying Microsoft Excel 2010. Funding This work was supported by the Hungarian Academy of Sciences [János Bolyai Research Scholarship grant No. BO/00751/16/8]; the National Research, Development and Innovation Fund [grant No. NKFI-6, K120383]; the EU-funded Hungarian grant [EFOP-3.6.1-16-2016–00008]; and the Ministry of Human Capacities [UNKP-17-4 New National Excellence Program to Z.K.]. Acknowledgments The authors thank Professor Aaron Chang (Kaohsiung Medical University, Graduate Institute of Natural Products, Kaohsiung, Taiwan) for the Astragalus membranaceus (Fisch.) Bunge seeds. We are also grateful to Dr. Attila Pécsváradi (Department of Plant Biology, University of Szeged) for his valuable advice and help. Disclosures The authors have no conflicts of interest to declare. References Arasimowicz-Jelonek M. , Floryszak-Wieczorek J. 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Google Scholar Crossref Search ADS PubMed Abbreviations Abbreviations BSA bovine serum albumin CAT catalase DAF-FM DA 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate DHE dihydroethidium DHR dihydrorhodamine 123 DPI diphenylene iodonium FDA fluorescein diacetate FITC fluorescein isothiocyanate GSNO S-nitrosoglutathione GSNOR S-nitrosoglutathione reductase H2O2 hydrogen peroxide NBT nitroblue tetrazolium NBT nitroblue tetrazolium NO nitric oxide NO2 nitrogen dioxide radical N2O3 dinitrogen-trioxide N2O4 dinitrogen tetroxide NOX NADPH oxidase NR nitrate reductase O2– superoxide OH hydroxyl radical ONOO– peroxynitrite PBS phosphate-buffered saline POD peroxidase RNS reactive nitrogen species ROS reactive oxygen species Se selenium SIN-1 3-morpholino-sydnonimine SMT selenocysteine methyltransferase SNO S-nitrosothiol SOD superoxide dismutase © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Plant and Cell PhysiologyOxford University Press

Published: Sep 1, 2018

References

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