1. Introduction
Selenium (Se) is present in the environment in elemental form or in the form of selenide (Se
2−), selenate (SeO
42−), or selenite (SeO
32−). It is widely distributed in the Earth’s crust at concentrations averaging 0.09 mg kg
−1 and in trace quantities in most plant and animal tissues [
1]. Selenium is not classified as an essential plant nutrient, but it is important for human and animal nutrition [
1,
2]. Plants are capable of Se accumulation and transformation of the toxic inorganic forms into different chemical species, and thus can be used for Se-phytoremediation [
3,
4]. Selenate uptake through the root plasma membrane is mediated by the high-affinity sulfate transporter encoded by the
SHST1,
SHST2, and
SHST3 genes [
5]. The expression of these root membrane transporters is negatively regulated by reduced glutathione (GSH) and/or sulfate (SO
42−). On the other hand, it is positively regulated by
O-acetylserine because the latter is a precursor of cysteine and a product of nitrate assimilation [
6]. The problems of transport, biochemistry, phytovolatilization, and phytoremediation of Se in higher plants have been reviewed by Terry and coworkers [
4]. More recent papers show the suitability of transgenic plants for hyperaccumulation of Se from the environment [
7–
9]. Meta-analysis can be also used to suggest strategies for remediation of soils contaminated by various elements, including selenium [
10].
Plants of the species
Astragalus,
Stanleya,
Morinda,
Neptunia,
Oonopsys, and
Xylorhiza are assumed to be Se-accumulators able to accumulate from hundreds to several thousands of mg of selenium per kilogram of dry weight. These plants grow readily on seleniferous soils. Crop plants and species of the genera
Distichlis or
Atriplex are Se-non-accumulators and grow on nonseleniferous soils (usually these can accumulate only 0.1 to 1 mg Se/kg dry weight, however maximum accumulated amounts of Se of about 25 mg Se/kg dry weight have been reported) [
4]. Additionally, plant species which are referred to as secondary Se-accumulators (
Astragalus,
Aster,
Castilleja,
Comandra,
Grayia,
Brassica and others) are plants growing on soils with low selenium content yet able to accumulate high concentrations of the element (1,000 mg Se/kg dry weight) [
11]. Moreover, has been shown that selenite contributes to a larger accumulation of non-protein thiol compounds in the roots and selenate contributes to their accumulation in the shoots [
12]. Reduced glutathione (GSH) belongs to the group of important non-protein -SH-rich molecules. This important antioxidant plays a role in the detoxification of a variety of electrophilic compounds and peroxides. The process is catalyzed by glutathione
S-transferases and glutathione peroxidase [
13,
14]. GSH is highly reactive and often found conjugated via its sulfhydryl moiety to other molecules such as NO (
S-nitrosoglutathione). In addition, it can be used for synthesis of phytochelatins [compounds with a basic formula (γ-Glu-Cys)
n-Gly (n = 2 to 11)] participating in the detoxification of heavy metals in plants. Phytochelatins have the ability to bind heavy metal ions via the SH groups of cysteine units and transport them to vacuole, where any immediate toxicity does not endanger the organism [
15–
19]. Plant cells display other ways to detoxify heavy metals in addition to phytochelatins [
20].
The nettle (
Urtica dioica L.) is a perennial plant growing in temperate and tropical wasteland areas around the world. The plant was naturalized in Brazil and other parts of South America. The maximum typical height of this plant species ranges from 2 to 4 meters. It produces pointed leaves and white to yellowish flowers. In folk medicine nettles have been used as a diuretic agent and to treat arthritis and rheumatism. Nowadays nettle is an important medical herb and consumed as a component of the human diet due to its content of minerals, chlorophyll, amino acids, lecithin, carotenoids, flavonoids, sterols, tannins and vitamins. Furthermore the roots of this plant contain other biological active compounds such as scopoletin, sterols, fatty acids, polysaccharides and isolectins. Several compounds present in the nettle have demonstrated antiviral properties potentially applicable for treatment of certain diseases e.g., HIV [
21], and several common respiratory viruses [
22]. Some compounds have also anti-proliferative effect on human prostate cancer cells [
23].
The aim of this study was to investigate the effect of selenium on the growth and the Se accumulation in the certain parts (roots, leaves, stamp and apex) of nettle plants and the possible mechanism(s) of Se transport Furthermore, a possible mechanism of Se transport was suggested.
2. Experimental Section
2.1. Chemicals
Sodium selenite and other analytical grade reagents were purchased from Sigma Aldrich Chemical Corp. (St. Louis, MO, USA). Water used for preparation of solutions was demineralised by reverse osmosis using Aqua Osmotic 02 (Aqua Osmotic, Tišnov, Czech Republic) and further purified by Millipore RG (18 MΩ Millipore Corp., Billerica, MA, USA). Standard 10 mg/mL stock solutions of selenium were prepared by dissolving sodium selenate in water and stored in dark at 4 °C. The working standard solutions were prepared daily by dilution of the stock solutions.
2.2. Plant Material and Cultivation
Grown-up plants of the dioecious nettle (
Urtica dioica L.) were used as a primary plant material. Nettle plants were removed from the soil and then green above-ground parts of the individual plants were cut and remaining rhizomes were rinsed with water. Twelve cuttings (2–3 centimeters long) were prepared from the rhizome and the surface of each cut was treated with a growth stimulator (AS–1, 0.06% nicotinic acid; 0.06% potassium α-naphtholacetate, Czech Republic). Modified cuts were planted in a mixture of garden soil and coarser river sand (1:1) for 20 days. After the root system developed (as soon as the first leaves appeared above the soil surface) the plants were singly placed in prepared plant boxes. River sand (3–4 cm) and 2,100 g of a mixture of garden soil and coarser river sand (1:1) were deposited in layers into each plastic plant box (15 cm × 15 cm × 15 cm). The plant boxes with young plants were placed in free space in an aviary covered with polyethylene sheet. Garden substrates used in this study were purchased from AGRO a.s. (Česká Skalice, Czech Republic). The substrate contained following essential nutrients: 275 mg N, 165 mg P
2O
5 and 425 mg K
2O per litre. Declared pH value of the garden soil aqueous extract was 6. After three days acclimatization of the replanted plants, 5 mL (2 mg Se) or 10 mL (4 mg Se) Na
2SeO
3 were applied to each plant box according the scheme shown in
Table 1. Adequate soil wetness was maintained by periodic watering. After 77 days (from selenium application) plants were carefully removed from the plant boxes and their above-ground and root parts were separated. The above-ground part was divided into an apex, younger leaves (first ten leaves from the apex), older leaves and a stalk. Underground parts were carefully cleaned of soil and washed on a sieve with flowing tap water and then with distilled water and placed on a filter paper to dry.
2.3. Sample Preparation for Fresh and Dry Weight Analysis
The plants were weighed on Sartorius R160P balances (Sartorius GmbH, Goettingen, Germany) immediately after collecting from the filter paper. Plant material was dried to the constant weight with a Premed evaporator (KBC G-100/250, Warszawa, Poland) and weighed. Dried samples were milled to a fine-grained powder (Ika A11 basic, Germany).
2.4. Sample Preparation for Selenium Determination
Mineralization of samples (0.01–0.30 g of the plant powder) was done using a MSL 1,200 microwave kiln (Milestone, Italy). Five millilitres of 65% nitric acid (max. 0.02 ppm Se) and one millilitre of 31% hydroperoxide were added to the plant powder. The plant powder was digested by an ETHOS SEL microwave digestion furnace (Milestone S.r.l, Italy) using a MDR 300/10 module. Decomposition was run according to the following program: 00:00–01:00 min, 250 W; 01:00–03:00 min, 0 W; 03:00–08:00 min, 250 W; 08:00–13:00 min, 400 W; 13:00–16:00 min, 500 W. Mineralized plant material was quantitatively transferred into the volumetric flasks and diluted by water up to10 mL.
2.5. Determination of Selenium Using Graphite Furnace Atomic Absorption Spectrometry
A UNICAM series M atomic absorption spectrometer with a graphite cuvette heated by an electric resistance coupled with a FS 95 autosampler (UNICAM) was used for selenium analysis. Ni(NO3)2 was used as a modifier. Zeeman correction in combination with correction via deuterium discharge lamp was used for background correction D2. The wavelength used (196.0 nm) was separated out from selenium lamp light to get the highest sensitivity.
2.6. Preparation of Plant Tissues for Thiol Determinations
Weighed plant tissues (approximately 0.2 g) were transferred to a test-tube and prepared according to Supalkova
et al. [
24]. Then, liquid nitrogen was added to the test-tube, and the samples were frozen to disrupt the cells. Subsequently, 1,000 μL of 0.2 M phosphate buffer (pH 7.2) was added to the test-tube. The mixture was prepared using an ULTRA-TURRAX T8 hand-operated homogenizer (IKA, Germany) at 25,000 rpm for 3 minutes. The homogenate was transferred to a new test-tube. The homogenate was shaken on a Vortex-2 Genie (Scientific Industries, New York, NY, USA) at 4 °C for 30 min. The homogenate was centrifuged (14,000
g) for 30 min at 4 °C using a Universal 32 R centrifuge (Hettich-Zentrifugen GmbH, Tuttlingen, Germany). The supernatant was filtered through a membrane filter (0.45 μm nylon filter disk, Millipore, Billerica, MA, USA) prior to analysis.
2.7. Determination of Thiols
The high performance liquid chromatography with electrochemical detection (HPLC-ED) system consisted of two solvent delivery pumps operating in the range of 0.001–9.999 mL/min (Model 582 ESA Inc., Chelmsford, MA, USA), a Metachem Polaris C18A reverse-phase column (150.0 × 2.1 mm, 5 μm particle size; Varian Inc., Santa Clara, CA, USA) and a CoulArray electrochemical detector (Model 5600A, ESA). The electrochemical detector includes three flow cells (Model 6210, ESA). Each cell consists of four analytical cells containing a carbon porous working electrode, two auxiliary and two reference electrodes. Both the detector and the reaction coil/column were thermostated. The optimal conditions were as follows: a gradient profile for simultaneous thiol separation starting at 100:0 (80 mM TFA-methanol), kept constant for 9 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 18 to 19 min, mobile phase flow rate of 0.8 mL/min, and column temperature of 40 °C. The sample (5 μL) was injected using an autosampler (Model 540 Microtiter HPLC, ESA). Further details are described in Diopan
et al. [
25].
2.8. Descriptive Statistics
Data were processed using MICROSOFT EXCEL® (USA) and STATISTICA.CZ Version 8.0 (Czech Republic). Results are expressed as mean ± standard deviation (S.D.) unless noted otherwise (EXCEL®). Statistical significances of the differences between Se content in treated and control plants were determined using STATISTICA.CZ. Differences with p < 0.05 were considered significant and were determined by using of one way ANOVA test (particularly Scheffe test), which was applied for means comparison.