Application of 2-Iminoselenazolidin-4-Ones (ISeA) for Beta vulgaris L. and Brassica rapa L. Plants Se-Biofortification

Abstract

Due to widespread selenium deficiency in food the aim of this study was to evaluate the effectiveness of a new Se(II)-containing organic chemical compound 2-iminoselenazolidin-4-ones (ISeA) in the form of a nanoscale associate (1–5 nm) solution for Swiss chard and komatsuna plants biofortification. Application of the chosen substance as a foliar treatment (2 mg·L⁻¹) and as an additive to a hydroponic nutrient solution (10 mg·L⁻¹) was performed. Both cultures had a high level of Se absorption, distribution and accumulation in leaves two or more times greater than in petioles. Se content in chard petioles (15 mg·L⁻¹) when applying ISeA as a component of the nutrient solution exceeded the accumulation of Se during foliar treatment (9.6 mg·L⁻¹) and the same trend in the komatsuna leaves was observed. When applying ISeA to the nutrient solution, an increase in komatsuna and chard biomass was seen at 36 and 68% and for leaf treatment by 21 and 45%, respectively. For komatsuna and chard an increase in the ratio of dry to fresh weight was also observed to be 27 and 26%, and for foliar treatment—0 and $16\%$, respectively. Treatments led to increase in chard plants height (7–17%), enlargement of leaves (19–42%), a rise in photosynthetic pigments (20–60%) and anthocyanin (2.9 and 2.2 times) concentration, and for komatsuna—the multiplication of leaves number ($28\%$) and their surface area (27–29%) as well as a rise in the concentration of anthocyanin (1.0 and 1.6 times) with foliar treatment and nutrient solution enrichment.

1. Introduction

Selenium (Se) is one of the key micronutrients that is necessary for normal functioning of the human and animal body. Selenium deficiency promotes the development of diseases with both specific and non-specific etiologies [1]. In the human body, a lack of selenium first of all causes heart pathology, decreased immunity, and a range of other pathological states, and special attention should be paid to infants as Se deficiency may result in mental retardation [2,3,4]. Selenium regulates the production of thyroxine and insulin in animal organisms. It is also an important component of 30 different selenoproteins and glutathione peroxidase enzymes necessary to fight free radicals, which is crucially important in cancer prevention. The element supports cell-protecting features that strengthen infection resistivity, takes part in DNA synthesis, preserves cognitive skills for the elderly, and it is involved in reproductive and other cycles [2,5].

The most effective way to prevent diseases caused by elemental deficiencies [6,7,8] is consumption of plant products enriched with Se. Practice has shown that taking medications, for example, in the form of sodium selenite, is highly likely to be associated with a high risk of exceeding permissible consumption doses and leads to subsequent toxic effects. Eating plant products rich in selenium, where it is delivered in an organically bound form, is the best choice for safe replenishment. Moreover, organically bound selenium is internalized 5–10 times better than mineral selenium by humans and animals [9].

Selenium enters the human body through chainlet “soil–harvest–human”, while the best biomarker for selenium deficiency in humans is the state of nails, hair, and blood [10]. Most of the natural soils engaged in agricultural production are poor in selenium, and its deficiency is observed in the daily diet of a significant part of the population [11]. Selenium deficiency is observed in more than 40 countries with an overall population of about 1 billion people according to WHO data, including Finland, China, Germany, New Zealand, Oceania, and the USA [12,13], and in Russia this deficiency is observed in 27 regions [14]. Thereupon, agronomic Se fortification has become the object of intensified scientific research and elaboration, and in Russia close attention has been paid to this problem [6,15,16,17]. Issues regarding crop bio-enrichment with selenium have become widely discussed and explored in the last 10 years (Figure 1), according to the PubMed database content.

Most of the published information is devoted to studies of inorganic forms of selenium—most often in the form of sodium or barium sals as selenate Se (VI) and selenite Se (VI) [18,19,20,21]. These forms are soluble in water and thus in soil or substrate. The use of inorganic forms of selenium in fertilizers has important features, as many authors of published works have mentioned. The solubility, mobility, and bioavailability of Se in soil usually depend on its exact chemical form and on its coupling with soil particles [21,22,23,24]. Selenate (i.e., Se with an oxidation rate +6) is a more effective form than selenite (i.e., Se with oxidation rate +4) to use in soil fertilization at neutral and alkaline pHs [25,26]. On the other hand, selenate is easily washed out of the soil solution, and its absorption can be reduced due to the presence of competing ions such as K⁺, Ca²⁺, Mg²⁺, ${\mathrm{SO}}_3^{2-}$, and Cl⁻ [22]. In acid conditions with a high clay content, selenite is the most common form of Se in soil [23,27]. This form is also rapidly absorbed by plants, but selenite establishes strong bonds with metal oxides or soil organic matter, which leads to low bioavailability for plants. Only about $12\%$ of the Se introduced directly into the soil is restored by plants; the amount can be higher with foliar treatment [28].

At the same time, a number of studies have noted that organic Se(II) complexes such as selenium–amino acids have exceptional accessibility for plants [29,30]. Thus, Selenocysteine (SeCys) and selenomethionine (SeMet) were absorbed by rape and wheat plants 2–20 and 40–100 times faster, respectively, when compared to selenates or selenites [30]. These compounds are suggested to enter plant cells through amino acid transporters, as nowadays there are many classes of the discovered examples [31]. After extensive analysis of the above-mentioned factors, it was assumed that synthetic organic compounds of Se(II) should be of scientific interest to discover and research, as these substances could successfully prove themselves as an effective Se-fortification agent.

Attention has been drawn to the organic form of sulfur (S)—thiourea—to form some working hypothesis, as it is the closest analogue of Se in terms of atomic structure and chemical properties [32]. Theoretically, selenourea should exhibit properties close to a sulfur-containing analog. This compound is chemically unstable under normal conditions and irradiation, and is decomposed to an elementary Se form [33]. Therefore, it was decided to use it as a new source of selenium for the biofortification of a stable organic Se compound, which is a derivant of selenourea—2-iminoselenazolidine-4-ones (ISeA) [34], which belongs to the group of nitrogenous compounds with 2 nitrogen atoms and contains Se(II) in its structure. The substance is highly soluble in water and does not favor additional antagonistic reactions with ions of nutrient solutions that provoke remnant falldown. The supposed effectiveness of this substance for Se fortification has promising prospects.

Selenium from food products is absorbed by the human organism and this process is much more efficient when the source is organic (up to $95\%$) than non-organic (only up to $50\%$) [35]. That is why recently more and more attention has been paid to Se fortification for agricultural products, including widely consumed vegetables and greenery. Unlike cereals, these types of crops do not contain gluten and, therefore, are recommended for people with intolerances to constituent proteins. Green crops are relatively safe to consume as they do not accumulate selenium in harmful quantities. According to published data, for almost all agricultural plants, Se enrichment leads to a significant amounts of SeMet formation, while vegetable crops, along with SeMet, also intensively synthesize methylated forms, which can cause pronounced anti-carcinogenic effects [36,37]. This may become important with an increase in green and vegetable crops produced in industrial groundless cultivation systems at hydroponic facilities [38].

The latest research shows the prospects of Se-enriched green plants of the genera $Beta$L. and $Brassica$L. When growing chard in a floating system with a nutrient solution enriched with selenite (0, 1.0, 3.0, 5.0 mg·L⁻¹), an increase in the concentration of this micronutrient in leaves was observed and an optimum concentration (1 mg·L⁻¹) contributed to an increase in yield and chlorophyll content in the leaves [39]. It was found that Se nanoparticles (SeNPs), compared with selenate and selenite, have higher bioavailability with lower toxicity and are able to stimulate crop growth and synthesis of secondary metabolites [40,41,42].

It was shown that Se nanoparticles ENMs with a negative charge (Se (–)) had the highest Se content in the shoots of $Brassicachinensis$L. (3.7-folds) [43] and that a positive impact of both nanoparticles SeNPs and ${\mathrm{SeO}}_3^{2-}$ is manifested on $Brassicanapus$L. growth under Cd-stress conditions, reducing oxidative damage to proteins and membrane lipids [44]. The use of SeNPs in the cultivation of celery ($Apiumgraveolens$L.) for greens increased the antioxidant capacity by $47\%$ and the total flavonoid amount by $50\%$ [41].

The content of Selenium in crops can decrease with higher rates of fertilization, along with yield decrease, while low doses can stimulate growth, increasing yields [45]. Therefore, planning Se application methods for the biofortification of crops still remains an important issue for the building principles of “precision farming” [46]. Thus, the purpose of our research was to evaluate the effectiveness of the new selenium-containing organic compound ISeA used in the form of nanoscale associates for the biofortification of chard ($Betavulgaris$subsp.$vulgaris$var.$vulgaris$) and komatsuna ($Brassicarapa$ var.$perviridis$) plants via foliar treatment and via the addition of a nutrient hydroponic solution.

2. Materials and Methods

2.1. Plant Material

Two green crops belonging to two different botanical families were chosen as our objects of research—chard ($Betavulgaris$ subsp. $vulgaris$ var.$vulgaris$) variety ‘Pomegranate ’belonging to the Amaranth family ($Amaranthaceae$Juss.) and komatsuna or Japanese mustard spinach ($Brassicarapa$ var.$perviridis$) belonging to the cabbage family ($Brassicaceae$).

Chard of the ‘Pomegranate’ variety has the following variety characteristics: medium-ripened (from full germination to harvest in 30–40 days), leaf rosette is compact, petioles are elongated, bright red, and juicy; the leaves are bubbly, bright green, shiny with red veins; and the variety is resistant to florescence. The seeds were provided by the “Gavrish” company (Moscow, Russia).

Komatsuna or Japanese mustard spinach is an early-ripe green leafy vegetable (from germination to harvest 28–32 days); leaf rosette is erect, 18–20 cm high, and consists of 4–7 juicy, red-maroon whole oval smooth leaves; the taste is excellent, delicate, practically without bitterness; and the plant is moisture-loving, cold-resistant, and resistant to premature stalking. The seeds purchased from the “Semko” company (Moscow, Russia).

2.2. ISeA Obtaining

A sample of 2-iminoselenazolidine-4-ones (ISeA) was synthesized and provided by the laboratory of technology of complexones and complex compounds of the Kurchatov Institute Research Center (Kurchatov Complex of Chemical Research division).

A method to obtain ISeA was adopted on the basis of the interactions between selenourea and monochloroacetic acid [34]. The starting materials were suspended in methanol and kept at room temperature for 4 h. The process was carried out in the absence of light in an inert gas flow (nitrogen, argon). The reaction scheme is shown in Figure 2.

Upon cooling, the reaction solution was filtered, methanol was distilled, and the dry residue was purified by recrystallization from an isopropanol–ethyl acetate mixture. The target product was isolated when the system was cooled to a temperature of 2–4 °C, then filtered, washed with methanol, and dried. The final product, ISeA, was a cream-colored solid substance with the presence of crystal particles. The product yield after synthesis was $50\%$.

2.3. ISeA Particles Characterization

The morphology of the obtained particles was studied by transmission electron microscope (TEM) [47] images analysis. A transmission electron microscope Libra 200 FE HR (Germany) was used to obtain TEM images with the required quality. Microscopic gold meshes were used to prepare the TEM microscopy sample. Element analysis was performed by energy dispersive X-ray (EDX) spectroscopy, by means of a built-in energy dispersion spectrometer INCA X-STREAM2 (UK) operating at an accelerating voltage of 1 kV, incorporated with TEM.

2.4. Plant Cultivation Conditions

To prepare a nutrient solution for hydroponic plant cultivation, a set of FloraSeries (GHE, France) fertilizers was used. This medium contains all the necessary macro- and microelements in the component proportions recommended by the manufacturer for the vegetative growth of green crops. The nutrient solution contained the following concentrations of macro- and microelements: N-NO₃—9.64 mM; N-NH₄—1.07 mM; P-PO₄ 1.00 mM; K 5.77 mM; Ca 2.00 mM; Mg 1.65 mM; S-SO₄—1.75 mM; Fe 15.00 mM; B 20.00 mM; Cu 1.00 mM; Zn 5.00 mM; Mn 10.00 mM; and Mo 1.00 mM.

Cubes of mineral wool were used as a substrate. For root nutrition, ISeA was added to the working nutrient solution in the amount of 10 mg·L⁻¹. It is assumed that foliar treatment allows for more efficient use of Se fertilizers, reducing the burden on the environment [17,48]. Therefore, in our study, foliar treatment was carried out with an aqueous solution of ISeA at a concentration of 2 mg·L⁻¹. The treatments were carried out on the 30th day after germination (Figure 3).

Plants were cultivated on 3 racks of a tiered structure in a climatic room (maintaining the following growth parameters: day/night temperature 25/22 ± 1.0 °C; and relative humidity $55\pm 5\%$). The germination and growing conditions were equal for both species. Lamps consisting of white LEDs with a ratio of spectral ranges Red (R), Blue (B), Green (G), and Far Red (FR)—35R, 17B, 45G, and 3FR—were used to illuminate the plants, with a photosynthetic photon flux density (PPFD) of 180 $\mathsf{\mu }$M·s⁻¹·m⁻² (Figure 3). The light period duration was 16 h. The lighting spectrum was designed to replicate the solar spectrum proportions of the ranges and was selected to meet the requirements for the green crops’ optimum growth [49].

2.5. Biometric Indicators

On the 35th day after the union shoots emerged, the following biometric parameters were measured and evaluated: height, fresh and dry weight (FW and DW), proportion of dry matter, number of leaves, and leaf surface area. Ten plants were picked out from each group to make measurements. The FW and DW of plants were determined using a Sartorius LA230S Laboratory Scale (Germany). To determine DW, samples were exposed in an oven for 1 h at a temperature of 105 °C. The leaf area was determined on a photoplanimeter LI-COR LI-3100 AREA METER (USA).

2.6. Pigment Content

The quantitative analysis of the pigments was carried out by extracting from plant tissues with solvents. Five samples were taken from the second or third sheet from above for each variant of the experiment. The pigment extract absorbance was determined using a spectrophotometer SSP-705 (Specks, Russia) at wavelengths of 662, 644, and 440 nm. The concentration of chlorophyll a, b, and carotenoids was calculated using the Holm–Wettstein formula for $100\%$ acetone [50].

Anthocyanins were extracted with $1\%$ HCl from leaves (0.3 g). The absorption index of cyanidin-3.5-diglycoside in a $1\%$ solution of hydrochloric acid at 510 nm wavelength was used to evaluate the total anthocyanin content [51].

2.7. Sugar Content

The leaves of each experimental variant were placed in a mortar and ground until juice appeared, then this juice was analyzed using a $\stackrel{..}{\mathrm{M}}\mathrm{aster-alpha}\stackrel{..}{\mathrm{r}}\mathrm{efractometer}$ (ATAGO, Russia) to determine the sugar content in the samples. Five samples were selected from each variant of the experiment.

2.8. Element Analysis

An atomic emission spectrometer Thermo iCAP 6300 Duo (USA) was used to perform elemental analysis and determine Ca, K, Mg, Cu, Fe, Mo, and Se contents. Preliminary sample preparation was carried out using a microwave decomposition system Speedwave Entry (Berghof, Germany) in C.P. nitric acid. Three samples of 0.1 g of dry matter for each experimental variant from different parts of the leaves (petioles and leaves) were prepared to refine the element distribution. Samples were placed in Teflon containers with nitric acid (10 mL) for decomposition, then 1 mL of the solution sample was taken and diluted 10 and 100 times to determine micro- and macronutrients, respectively.

2.9. Statistical Analysis

To obtain reliable results the experiment was carried out in three repetitions. Subsequent statistical processing of measurement results and diagram plotting were carried out in MS Excel 2010. Two-factor analysis of variance (ANOVA) with a significant difference of $p<0.05$ was implied to determine significant results.

3. Results

3.1. Characterization of SeNPs

TEM images show that the resulting ISeA particles are large nanoaggregates (Figure 4a) consisting of smaller spherical molecular nanoassociates several nanometers (Figure 4b) in diameter. Selenium in the resulting particles is locked in a crystalline state, the diffraction pattern presented in Figure 4b demonstrates the presence of diffraction rings corresponds to the crystal lattice sizes of 3.7 Å, 2.2 Å, and 1.9 Å, which in turn correspond to the hexagonal modification of selenium with the directions of the crystallographic planes (100), (110), and (200), respectively.

The EDX spectrum of the obtained particles, presented in Figure 4c, indicates the presence of pure Se in its unoxidized state. Distribution of the particles depending on size is presented (Figure 4d) with a base of HRTEM images of the obtained molecular nanoassociates. The size of $90\%$ of the particles lies in a range from 1 to 5 nm and the maximum for particle distribution is 3 nm. The half-width of the distribution curve is about 1.5 nm.

3.2. Effect of ISeA and Processing Method on Plants Biometric Characteristics

The basic parameters of plant growth and development are as follows: height, fresh weight, dry matter content (%), number of leaves, and average area of leaf plates, which are presented in the

Table 1. Biometric parameters for the aboveground parts of the Swiss Chard and Komatsuna plants with the use of ISeA as a foliar treatment (Foliar) and nutrient solution (Solution) on the 35th day of cultivation. Values represent mean ± SE ($n=10$). The different letters indicate significant differences among treatments according to Duncan’s test ($p\le 0.05$).

ISeA Treatment	FW (g)	DW/FW (%)	Number of Leaves	Leaf Area (cm2)	Plant Height (cm)
			Chard
Control	$26.9\pm 5.4$ a	$8.2\pm 1.3$ a	1$0.1\pm 0.8$ a	$435.2\pm 103.7$ a	$26.1\pm 3.5$ a
Foliar	$39.1\pm 4.5$ b	$9.5\pm 0.7$ b	$10.1\pm 0.8$ a	$518.6\pm 104.2$ a	$28.1\pm 1.9$ ab
Solution	$45.1\pm 2.3$ b	$10.3\pm 1.6$ b	$10.4\pm 1.2$ a	$618.4\pm 131.0$ b	$30.4\pm 3.7$ b
			Komatsuna
Control	$33.0\pm 4.0$ a′	$7.1\pm 1.2$ a′	$8.6\pm 1.2$ a′	$517.5\pm 104.6$ a′	$20.7\pm 2.7$ a′
Foliar	$40.0\pm 4.4$ ab′	$7.6\pm 1.0$ a′	$11.0\pm 1.8$ b′	$669.3\pm 126.9$ b′	$19.2\pm 2.6$ a′
Solution	$44.7\pm 3.9$ b′	$9.0\pm 1.4$ b′	$11.1\pm 1.2$ b′	$655.1\pm 132.4$ b′	$19.3\pm 2.2$ a′

. Data obtained show that ISeA at the applied concentrations had significant stimulating effect on almost all the basic biometric parameters of the plants (Figure 5).

3.3. Effect of Enrichment Method and ISeA on the Biosynthesis of Photosynthetic Pigments and Anthocyanins

The recorded values of pigment concentrations confirmed the different reactions and susceptibility of the studied crops to the absorption and metabolism of ISeA (Figure 6). Application of ISeA in any form had virtually no effect on the levels of chlorophyll a, b, and carotene in the Komatsuna samples. A completely opposite pattern was discovered for chard. With the help of ISeA fortification, the level of chlorophyll a and b in plant tissues increased, respectively, by 47 and $59\%$ after foliar treatment and by $21\%$ and $22\%$ after nutrient solution enrichment (Figure 6). The same trend was observed for carotenoid content: 45 and $13\%$ (after foliar treatment and nutrient solution, respectively).

ISeA fortification of chard plants had an even more pronounced effect on the synthesis of anthocyanins, which have antioxidant properties. Total concentration of the pigment varied significantly among the experimental variants (Figure 7). High variability of this trait is due to the uneven coloring of the leaves of both species. For chard plants, application of ISeA increased anthocyanin content by 2.9 and 2.2 times during foliar treatment and solution application, respectively, indicating little stress caused by the treatment. In komatsuna plants, foliar treatment with ISeA did not affect the anthocyanin content, and ISeA addition to the nutrient solution increased concentration by 1.6 times.

3.4. Effect of Enrichment Method and ISeA on Sugar Content

The ISeA addition also had a noticeable effect on the accumulation of sugars in both crops (Figure 8). Leaf treatment increased the sugar content in chard plants by 1.7 times, and addition to the nutrient solution by 2.1 times. In komatsuna plants, the effect of the treatments was not as pronounced (increase by 1.2, 1.1 times) as in chard plants.

3.5. Impact of ISeA and Enrichment Method on the Absorption and Accumulation of Se and Other Elements

Chard and komatsuna plants were selected based on previously conducted studies on crops of related species of the same botanical families [39,43,44] showed high biofortification results. Se accumulation in both crops occurred more in the leaf tissues than in the petioles (

Table 2. Content of Ca, K, and Mg in the aboveground organs of Chard and Komatsuna (g·kg⁻¹) DW. Letters signify the following variants: in ISeA treatment N—stands for control, i.e., absence of treatment; F—for foliar treatment; S—for ISeA addition to nutrient solution, taking into account parts of plants; P—stands for petioles; and L—for leaves. The different letters indicate significant differences among treatments according to Duncan’s test ($p\le 0.05$).

Treatment	Part of Plant	Ca	K	Mg
			Chard
Control	P	$86.2\pm 5.1$ a	$1129.2\pm 171.2$ b	$522.2\pm 13.2$ b
	L	$138.9\pm 21.1$ b	$728.6\pm 90.5$ a	$809.6\pm 57.3$ d
Foliar	P	$88.3\pm 3.9$ a	$779.8\pm 76.1$ a	$365.6\pm 76.1$ a
	L	$117.8\pm 9.3$ b	$869.5\pm 111.1$ ab	$598.0\pm 32.5$ c
Solution	P	$87.2\pm 14.0$ a	$999.3\pm 19.9$ b	$371.8\pm 29.9$ a
	L	$129.1\pm 10.6$ b	$1014.3\pm 79.8$ b	$605.0\pm 31.3$ c
			Komatsuna
Control	P	$89.4\pm 6.1$ a	$1045.8\pm 129.8$ d	$138.2\pm 16.9$ a
	L	$113.1\pm 13.3$ b	$527.0\pm 59.1$ ab	$212.8\pm 25.1$ b
Foliar	P	$119.4\pm 3.3$ b	$594.8\pm 17.4$ b	$118.2\pm 20.4$ a
	L	$137.2\pm 3.4$ c	$449.8\pm 86.5$ a	$170.3\pm 7.3$ b
Solution	P	$128.4\pm 14.1$ bc	$768.5\pm 89.7$ c	$200.2\pm 42.8$ b
	L	$124.1\pm 12.6$ bc	$308.3\pm 88.7$ a	$193.2\pm 31.7$ b

and

Table 3. Content of Se, Fe, Cu, and Mo in the aboveground organs of Chard and Komatsuna (mg·kg⁻¹) DW. Letters signify the following parts of plants: P stands for petioles and Lstands for leaves. The different letters indicate significant differences among treatments according to Duncan’s test ($p\le 0.05$).

Treatment	Part of Plant	Se	Fe	Cu	Mo
			Chard
Control	P	-	$250.2\pm 9.7$ b	$2.1\pm 0.1$ b	$1.5\pm 0.2$ a
	L	-	$297.1\pm 32.1$ b	$3.5\pm 0.5$ c	$5.8\pm 0.9$ c
Foliar	P	$9.6\pm 1.0$ a	$201.2\pm 24.3$ a	$0.9\pm 0.2$ a	$1.0\pm 0.5$ a
	L	$35.7\pm 7.4$ c	$248.1\pm 33.0$ ab	$2.3\pm 0.2$ b	$4.8\pm 1.5$ c
Solution	P	$15.0\pm 1.6$ b	$187.2\pm 12.0$ a	$1.9\pm 0.3$ b	$1.2\pm 0.8$ a
	L	$34.7\pm 9.4$ c	$235.3\pm 26.5$ ab	$2.0\pm 0.4$ b	$2.8\pm 0.2$ b
			Komatsuna
Control	P	-	$82.0\pm 1.9$ c	$1.3\pm 0.1$ a	$1.1\pm 0.2$ a
	L	-	$94.0\pm 8.7$ c	$1.6\pm 0.1$ a	$1.9\pm 0.1$ b
Foliar	P	$15.6\pm 1.1$ a	$50.7\pm 10.1$ a	$1.1\pm 0.1$ a	$0.9\pm 0.2$ a
	L	$31.1\pm 1.0$ c	$73.7\pm 4.7$ b	$1.6\pm 0.3$ a	$2.2\pm 0.6$ bc
Solution	P	$55.6\pm 0.3$ d	$86.0\pm 2.8$ c	$1.2\pm 0.1$ a	$2.5\pm 0.2$ c
	L	$23.8\pm 3.5$ b	$78.2\pm 4.7$ b	$0.9\pm 0.1$ a	$1.4\pm 0.6$ a

). In chard, the Se content in the plant leaves differed slightly depending on the method of ISeA application, and in petioles (the main part for food consumption) it was $56\%$ higher when added to the nutrient solution compared to foliar sprays Table 3. In both cases, for chard, high selenium concentrations were achieved: 9.6–15.0 mg·kg⁻¹ in petioles and 35–36 mg·kg⁻¹ DW in leaves. For komatsuna, there was little difference between treatments for both leaves and petioles, with the favor of adding to nutrient solution. Higher concentrations of the element in leaves (the main edible part) were achieved when using ISeA in the nutrient solution (by $79\%$) and in petioles by $53\%$ compared to the foliar treatment.

When analyzing the changes in a number of other nutritional elements in the samples of the Chard culture, it was found that the content of almost all the scanned chemical elements, except calcium and potassium, decreased slightly as a result of ISeA use (Table 2). There was an increase in K in leaves by $39\%$ when ISeA was added to the nutrient solution, while with foliar treatment there was a decrease in K in the petioles by $31\%$. Mg concentration in petioles and leaves decreased by an average of 25–30%; Fe concentration in petioles by 20–25%, in leaves—by 17–20%; Cu concentration decreased significantly in petioles only with leaf treatment by $57\%$, and in leaves by 34 and $42\%$ with leaf treatment and when added to the nutrient solution, respectively; and Mo concentration decreased significantly only in leaves when added to the nutrient solution by $51\%$ (Table 3).

Changes in the concentrations of other elements were also observed in ISeA-treated komatsuna plants, but at a different level. For example, in all treated experimental samples an increase in Ca concentration was observed compared to the control: in petioles by 34–44% and in leaves by 10–21% when treated with leaves and when added to the nutrient solution, respectively. There was also an increase in concentration of Mo in the leaves by $31\%$ when ISeA was added to the solution. Concentration of K decreased in leaves by 15 and $41\%$ and in petioles by 43 and $26\%$ with foliar treatment and addition to the nutrient solution, respectively. Fe concentration decreased only with leaf treatment in leaves by $21\%$, and in petioles by $36\%$. Mg concentration in plant leaves did not change significantly after treatment, but in petioles it increased by $31\%$ when ISeA was added to the nutrient solution. Cu concentration did not vary significantly. In general, it can be concluded that Se fortification by ISeA at the indicated concentrations caused a slight redistribution of other nutrients, but ensured effective selenium absorption by plant tissues.

4. Discussion

Despite numerous scientific studies on selenium bioenrichment strategies, the output of Se-enriched products suitable for human consumption remains a challenging objective. Different forms of selenium have different bioavailability, as well as different metabolic pathways in plants. Therefore, there is still insufficient comprehensive knowledge about the accumulation of Se by various agricultural crops, depending on element source form, dose of the substance, cultivation and processing methods, and other factors.

In our study, the use of nanoscale molecular associates of ISeA has led to encouraging results in several regards. Firstly, there was a clear positive effect on the physiological status of plants—a biomass increase. In addition, the concentrations of pigments (chlorophyll, carotenoids, and anthocyanins) in chard plants also increased significantly, which magnified the consumer value of the tested culture. Some published papers have also reported on the positive effect of Se fortification on plant development, photosynthesis, and antioxidant metabolism [39,52,53,54,55,56]. The closest to a reliable comparison of the results obtained can be carried out on examples of studies performed during the hydroponic cultivation of plants. Most studies look at Se fortification with inorganic forms: sodium selenate and selenite. Bio-enrichment of Swiss chard with selenium in the form of Na selinite at a concentration of 1 mg·L⁻¹ resulted in an increase in hydroponic crop yields by $20\%$, increase in chlorophyll content by $25\%$, while a reduction in DW by $37\%$ was observed, and high concentrations of introduced Se lead to lower yield gains [39]. There was also an increase in flavonoids content for chard, but the results were not statistically significant, unlike the present experiments. Impressive results were obtained: an increase in the chard’s biomass by $45\%$ and $68\%$, respectively, with foliar treatment of 2 mg·L⁻¹ and when adding 10 mg·L⁻¹ of ISeA to the nutrient solution (the increase in fresh biomass is proportional to the yield per area unit when cultivated in 1 tier). At the same time, in the experiments, an increase in dry weight was also observed with no tendency to decrease in biomass growth when boosting the ISeA concentration. An increase in the chlorophyll concentration in chard when applying ISeA to the nutrient solution was also equivalent to the considered experiment, despite a significant growth in the concentration of introduced Se, and foliar treatment showed itself to be more effective and raised the concentration of chlorophyll by an average of two times compared with the introduction into the solution.

When cultivating color-grained wheat 202w17, Se foliar treatment contributed to an increase in the anthocyanin concentration by $21.1\%$, while no difference was found for another variety with a lower content of anthocyanins [48]. In our studies, the introduction of ISeA in chard increased the anthocyanin content by 2.9 and 2.2 times during foliar treatment and introduction into solution, respectively. In komatsuna plants (the content of the pigment itself is lower for the species), foliar treatment of ISeA did not affect the content of anthocyanin, and addition to the nutrient solution increased its concentration by 1.6 times. It can be concluded that the richer the species/variety is in anthocyanins, the greater the effect of Se-fortification on this trait can be observed. It is also known that when growing corn under salinization conditions, foliar treatment of 20 mg·L⁻¹ sodium selenate contributed to the growth of biomass by 32–37%, chlorophyll a and b by 1.7 and more than 2 times, respectively [55]. Similarly, an increase in biomass (by $42.6\%$), plant height ($16.7\%$), phenolic content ($58.9\%$), and Se concentration in leaves ($22.2\%$) was observed in the study of Se fortification of 4 $\mathsf{\mu }$M·L⁻¹ sodium selenate of evidium ($Cichoriumendivia$ L. (var.$crispumHegi$)) in a hydroponic facility. At the same time, the Se concentrations in the leaves were 8.83 and 5.76 mg·L⁻¹ DW when enriched through a nutrient solution and foliar treatment, respectively [57].

The positive effect of Se treatment (0–30 $\mathsf{\mu }$M·L⁻¹ sodium selenite; 0.2–60 $\mathsf{\mu }$M·L⁻¹ sodium selenate) on the biometric parameters of lettuce ($Lactucasativa$L. var.$capitata$) and antioxidant production was also noted in the study of the effect of Se fortification on plant growth under thermal stress [58]. The concentration of photosynthetic pigments did not change significantly when selenate was applied, and when selenite was applied at concentrations of 6 $\mathsf{\mu }$M or more, it even decreased slightly (by 10–14%).

Se introduction before exposure prevented a decrease in biomass caused by thermal stress compared to the control variant by 31–38% and 30–48% for fresh and dry biomass, respectively. Also, the values of chlorophyll a and b concentrations decreased slower with Se treatment, and the concentration of carotenoids increased by $11\%$. The absorption of selenium by plant shoots with selenite usage was 3.7–30.6 mg·kg⁻¹ DW, with selenate addition up to 4.7–43.3 mg·kg⁻¹ DW. Positive results were obtained during the introduction of sodium selenate into a hydroponic solution at doses of 0.0–40.0 $\mathsf{\mu }$M·L⁻¹ when growing radishes ($Raphanussativus$L. cv. $Saxa$) [59]. Selenate treatment resulted in a 20–0% increase in biomass, with Se accumulation of 242 mg·kg⁻¹ DW in leaves and 85 mg·kg⁻¹ DW in plant roots. The authors supposed the most optimal dose of selenate application as 5–10 $\mathsf{\mu }$M·L⁻¹. Selenate was metabolized in radishes into the anticarcinogenic compound Se-methyl-selenocysteine. For $Brassicanapus$L. plants under Cd-stress conditions, increased biomass accumulation was also observed with use of selenite and SeNPs, also, Se nanoparticles had an increased antioxidant effect and demonstrated a wider range of useful concentrations [44]. In our experiment, komatsuna plants showed larger biomass growth rates (21–36%) compared to the mentioned representatives of the same genus, but this may be due to a combination of species characteristics and experimental conditions.

At the same time, some other studies have shown that Se biofortification under hydroponic conditions did not result in a significant change in plant growth indicators and content of biologically active substances, but provided an increase in the trace element concentrations in plant tissues [60,61,62]. In experiments with lettuce ($Lactucasativa$L. cv.$Venezaroxa$), when studying sodium selenate and selenite additives at dosages of 0–40 $\mathsf{\mu }$M·L⁻¹, high values of selenium content in shoots were recorded in the range of 23.2–50.8 mg·kg⁻¹ DW for sodium selenite and 57.4–602 mg·kg⁻¹ DW for sodium selenate [60]. The cultivation of basil ($Ocimumbasilicum$L. cv.$Tigullio$) with the addition of sodium selenate at concentrations of 0.5–2.0 mg·L⁻¹ was accompanied by the accumulation of trace elements to the level of 0.98–1.25 mg·kg⁻¹ DW in stems and 2–5 mg·kg⁻¹ DW in leaves [61]. Generally, in addition to the Se concentration increase, the use of the compound did not have other significant effects on the product quality indicators—biomass, total amount of phenols, chlorophyll, and carotenoids. The same trend was observed in spinach sprouts ($Spinaciaoleracea$L.) [62]. In that experiment, Se was also introduced as sodium selenate (0–5.2 $\mathsf{\mu }$M). The maximum selenium content in leaves was observed at a level of 9–11 mg·kg⁻¹ DW.

In the present study the reaction of plants to ISeA processing is in good correspondence with earlier published information. ISeA has demonstrated high levels of absorption, distribution, and accumulation in both of the considered methods of application—with a nutrient solution and foliar treatment. As for the effect of Se on the absorption of other nutrients, studies conducted over various crops do not provide a clear understanding of trends in concentrations change. Nutrients entry into plants is species-specific and depends on form of the substance used, application method of and cultivation conditions. It is known that addition of Se reduced Mg content in leaves and Ca and Mg content in stems of spinach ($Spinaciaoleracea$L. cv.$Missouri$) [62]. Results on changes in the concentration of Ca and K ions in leaves of komatsuna plants were obtained when growing spinach in hydroponics using sodium selenite at a dose of 1–10 mg·L⁻¹ [63], which is in accordance with the results outlined above.

According to the information provided, Ca content increased in shoots by 69–81%. Potassium content in shoots and leaves decreased when a high concentration of Se was introduced in the solution (more than 4 mg·L⁻¹) by 16–31%. It was also found that selenate promotes absorption of Mo and a decrease in K content, and selenite reduces the accumulation of Mo, Cu, and Fe, but contributes to an increase in the concentration of Ca and Mg in lettuce [60]. When cultivating color-grained wheat, Se foliar treatment also contributed to an increase in Ca and Mg concentrations [48]. When studying Se fortification with sodium selenite (0.0, 1.0, 2.0, 4.0 and 8.0 $\mathsf{\mu }$M·L⁻¹) of endivium ($Cichoriumendivia$) plants, concentration of K and Mg were found to show no significant change, however, a noticeable decrease in Ca was noted at concentrations of Se 2–8 $\mathsf{\mu }$M·L⁻¹ (by 8–32%) [57].

Despite the general tendency to decrease potassium during Se fortification, under salinization conditions, maize leaf treatment with selenate increased concentration of K, improving K⁺/Na⁺ ratio, which led to a positive effect over plant growth [55]. In our experiments, different reactions of various plants species to the types of ISeA treatment to absorption of K were also observed. Thus, in komatsuna plants, there was a decrease in the K accumulation during leaf treatment by 15 and $43\%$ and during application to nutrient solution by 41 and $26\%$ (in leaves and petioles, respectively). As for chard, K concentration depended on the method of ISeA application: a concentration increase in leaves when applied to a nutrient solution and a decrease in petioles during foliar treatment. The absorption of Ca also had a species-specific character: no significant changes in concentration were found for chard, and komatsuna accumulated the element in both its leaves and petioles. It was previously noted that SeNPs are involved in maintaining intracellular calcium homeostasis, contributing to the formation of disulfide bonds and the restoration of the waxy outer leaf surface layer [44], which may partially explain the results obtained.

Analyses of the applied concentrations ranges of selenium-containing fertilizers in hydroponic nutrient solutions showed a wide range of effective doses depending on the culture and experimental conditions from 0.5–12 mg·L⁻¹ (0.5–60 $\mathsf{\mu }$M) [57,59,62,63]. In our study, experimental plants of both crops reacted positively to the introduced doses of ISeA both in the nutrient solution (10 mg·L⁻¹) and during foliar treatment (2 mg·L⁻¹) without any signs of intoxication. Other published sources contain data on the effectiveness of much lower concentrations of Se additives, for example, when growing spinach plants using sodium selenate in a solution at concentrations of 2.6 and 3.9 $\mathsf{\mu }$M (0.5 and 0.7 mg·L⁻¹), that led to the accumulation of Se 9–11 $\mathsf{\mu }$g/g FW. The concentration of sugars also increased, but the results were not statistically significant [62]. In almost all publications, a linear dependence of the selenium concentration in plant organs on the dose of the applied fertilizer was proposed. However, the best biometric indicators (biomass, plant growth, number of leaves, etc.) and important metabolites (chlorophyll, phenolic complex) were recorded at medium Se doses, which did not lead to a toxic effect and inhibition of growth: 4 $\mathsf{\mu }$M [57], 20 $\mathsf{\mu }$M [59], 1 mg·L⁻¹ [63]. A wide range of initial doses of Se fertilizers was also tested on various crops [17,64,65] in the case of leaf treatment method usage, the success of which has been proven. It is assumed that Se nanoparticles have a wider range of useful concentrations [44]. Moreover, their size affects the Se transfer coefficient. Thus, when studying the absorption of SeNPs with sizes of 50, 100, and 150 nm in wheat and rice, it was found that they more easily pass into the plant in the form of small particles (50 nm) at low pH values (3.5) [65]. Using the example of rice plants, it was also found that chemosynthesized selenite SeNPs with a size of 86.1 nm, unlike selenate, quickly transformed into organic forms in plants with a predominance of SeMet [66]. The use of chitosan-modified Se (Ch-SeNP) nanoparticles on radish cultures of $Raphanussativus$ L. and Sareptskaya mustard ($Brassicajuncea$(L.) $Czern.$) confirmed biotransformation of Ch-SeNPs into selenoaminoacids: SeMet, semethylselenocysteine (SeMetSeCys), and $\gamma$-glutamyl-Se-MetSeCys [67].

The molecular weight of ISeA—169 is lower than the molecular weight of sodium selenate and selenite. It is assumed that due to the nanoscale molecular associates and its organic nature, the compound is well absorbed and transported similarly to other known synthetic ligands from the class of aminopolycarboxylic acids (EDTA, DTPA, etc.), which are successfully used for chelating trace elements and heavy metals in fertigation and phytoextraction issues [68,69,70]. However, we cannot predict how the absorption of ISeA by a plant cell takes place—in the form of nanoscale associates of molecules or by individual molecules formed after the decay of associates during cell wall adsorption. Also, at this moment any serious data on the possible pathways of ISeA metabolism by plants are missing. By indirect assumption ISeA can transform into selenoaminoacids, so follow-up studies are to be devoted to this issue.

5. Conclusions

ISeA use as a source of selenium for plants has led to very good results for both tested leaf crops, especially for chard. This opens up great opportunities for the practical use of this composition. A significant positive effect is observed not only in terms of Se accumulation, but also in terms of increase in chard and komatsuna plants biomass, an improvement in their gustatory (accumulation of sugars) and consumptive (accumulation of anthocyanins) qualities. Nevertheless, the authors of this study are well aware that there is still a very long way to go in the scientific research before we can understand the mechanisms and scope of acceptable/effective concentrations of ISeA. First of all, it is necessary to study the range of lower concentrations both in the nutrient solution and, especially, during spraying and we need to perform a direct screening comparative study with known inorganic Se substances—sodium selenate and selenite. It is also obvious that a future study program should include expansion of tested crops and of ISeA use conditions, including soil and soilless methods of cultivation, and also considering the plant growth phase. And, of course, an important point is to clarify Se distribution and speciation inside plants as a result of ISeA absorption: the forms in which it transforms and plant locations where it accumulates. Answers to these questions will allow us to develop accurate Se biofortification programs and make the best use of the advantages of the proposed compound.