Effect of Foliar Application of Sodium Selenate on Mineral Relationships in Brassicaceae Crops

Abstract

The relationships of selenium (Se) with other elements in plants is important for producing functional food with high Se contents and a predicted quality. To unveil the peculiarities of the element interactions, eight botanical varieties of Brassica oleracea L. were grown in similar conditions with or without foliar application of sodium selenate. High varietal differences, elicited by the Se supply, were recorded with regard to the accumulation of the elements examined, except for Mg, P and Si. Cabbage florets (broccoli and cauliflower) were characterized by both the lowest total mineral content and number of elements showing content changes under the Se supply (7–8 out of 25), whereas in Savoy cabbage, the highest number of minerals displayed content changes (13–14 from 25). The Se treatment did not significantly interfere with the high correlation coefficients recorded between Sr–Ca, Co–Ni and Zn–Mg (0.824–0.952). The selenium biofortification value varied from 12 to 138 depending on the species and was inversely correlated with the Si accumulation in the control plants (r = −0.872, p < 0.001). A significant decrease in the correlation coefficients occurred due to the Se supply regarding Zn with P and Co, Ca with Co and Li, Li and V, and Na and Sn, while the V–Pb relationship was significantly enhanced. Among the 25 elements studied, Cr demonstrated the highest number of significant correlation coefficient changes (with K, Na, P, Si, Zn, Cu, Co, I, As, Pb, and V). The results of this research prove the variability of the element interactions under foliar Se treatments in Brassica oleracea plants and reveal, for the first time, an inverse correlation between the Se biofortification level and Si content in untreated plants.

1. Introduction

The biofortification of Brassica oleracea plants with Se provides wide possibilities to produce functional food with high anticarcinogenic activity, which is capable of improving the human Se status. Due to significant sulfur (S) accumulation, these plants easily replace S with its chemical analog Se [1,2], forming Se-containing amino acids and peptides and their methylated forms (Se-MeSeCys, γ-Glutamyl-Se-Me-SeCys) [2,3]. Recent investigations also revealed the formation of glucosinolate Se derivatives [3,4,5]. All these Se compounds demonstrate higher protection effects against cancer when compared to non-methylated Se-amino acids and non-substituted glucosinolates, modulating the immune status of human organisms and protecting the latter against cardiovascular and viral diseases [6].

Taking into account the popularity of Brassica oleracea plants in the human diet, attempts have been made to develop appropriate technologies for Se biofortification of the following botanical varieties: kohlrabi [7]; white [8], red [9] and Savoy cabbages [10]; broccoli [11]; Indian mustard [12] and Chinese cabbage [13], paying special attention to the increase in secondary metabolites’ accumulation.

The difficulty in Brassicaceae species biofortification relates to the variable Se accumulation abilities, ranging from non-accumulator to Se-hyperaccumulator species [1,2,14,15], though most Brassica oleracea plants demonstrate a high ability to absorb, metabolize and concentrate high amounts of Se in their tissues [6].

Another significant issue is represented by the rather fragmented understanding of Se’s interaction with other elements [16,17] and especially of Se’s effect on the interaction changes between other elements. A general approach to this uncertainty demonstrates several possible effects: changes in the soil characteristics and soil microbial diversity; regulation of the genes involved in the elements’ transportation; antioxidant defense improvement; and the effects on the uptake and transport of several elements (Na, B, Cl, Mo, Ni, Cu, Fe, Mn and Zn) [16]. Many investigations have been devoted to plants’ protection against heavy metals via Se biofortification [18,19]. Different reports suggest the possible beneficial effects of Se supply on macro- and micro-element accumulation [20]. Highly significant interactions between Mn, Fe, Co, Cr and V were revealed in chervil fortified with Se [21]. The antagonistic effect of Se on I accumulation was recorded in kohlrabi leaves [7], while a synergistic effect of Se and I was shown in Brassica juncea [12]. Plenty of factors affecting the Se–minerals interaction, which are connected with different Se forms’ utilization [9], various methods of Se supply [22,23] and genetic peculiarities [23], lead to complex and inconclusive results. Furthermore, up to the present date, no attempts of a broad comparison between different B. oleracea representatives grown in similar conditions have been carried out. In this respect, the high variability of the mentioned results entails the need for a deeper investigation into the Brassicaceae species, especially the botanical varieties of Brassica oleracea, to show the high nutritional importance and the ability to tolerate increased levels of Se. We hypothesized that, in case of the absence of a direct Se effect on the soil’s characteristics, Se supplementation might significantly change the plant’s elemental status via modulation of the relationships between different elements. To verify this hypothesis, eight Brassica oleracea botanical varieties were grown in similar conditions and foliar-treated with the sodium selenate solution.

2. Material and Methods

2.1. Growing Conditions and Experimental Protocol

This research was conducted in 2021 and 2022, from April to September, at the experimental fields of the Federal Scientific Vegetable Center, Moscow region, Russia (55°39.51′ N, 37°12.23′ E). A loam sod-podzolic soil was used (The podsol reference soil group according to [24]) with the following initial soil characteristics: pH 6.2; 2.12% organic matter; 1.32 mg-eq 100 g⁻¹ hydrolytic acidity; 18.5 mg kg⁻¹ mineral nitrogen; 21.3 mg kg⁻¹ ammonium nitrogen, the sum of the absorbed bases by as much as 93.6%; 402 mg kg⁻¹ mobile phosphorous; 198 mg kg⁻¹ exchangeable potassium, 1 mg kg⁻¹ S; 10.95 mg kg⁻¹ Ca; 2.05 mg kg⁻¹ Zn; 0.86 mg kg⁻¹ B; 220 µg kg⁻¹ d.w. Se; 7.65 mg kg⁻¹ Ni; 0.22 mg kg⁻¹ Cd; 1.6 mg kg⁻¹ As; 12.85 mg kg⁻¹ Pb. The soil quality was assessed using the certified methods described in the agrochemical workshop [25], with the soil mineral composition determination using an AAS Shimadzu GFA-7000 spectrophotometer (Shimadzu, Kioto, Japan).

The sowing was performed on 28 April in multicell containers, and the seedling transplant occurred on 3 June, using a plant density of 2.9 plants per m² (50 × 70 cm).

The values of the mean temperature and relative humidity (

Table 1. Monthly temperature and precipitation in 2021 and 2022.

	2021		2022
Month	Mean Temperature (°C)	Rainfall (mm)	Mean Temperature (°C)	Rainfall (mm)
May	13.8	81	10.0	55.5
June	21.8	20	18.6	24.6
July	22.0	38	20.2	66.1
August	19.4	36	22.3	13.7
September	9.1	58	9.6	125.7

) were recorded during the crop cycles by the meteorological station located at the experimental fields of the Federal Scientific Vegetable Center.

The effects of Se biofortification on the mineral element composition of eight cultivars belonging to six cabbage botanical varieties, such as kohlrabi, broccoli, white and red cabbage, Savoy cabbage and cauliflower, were assessed.

Table 2. Brassica oleracea botanical varieties used and dates of sodium selenate treatment (50 mg L⁻¹) and plant harvesting.

Species	Cultivar	Plant Phenological Phase	Date of Foliar Sodium Selenate Treatment	Harvesting Date
Kohlrabi (Brassica oleracea var. gongylodes)	Dobrynya F1	Stem formation	(1) 01/07 (2) 11/07	24/07
Broccoli (Brassica oleracea var. italica)	Tonus	Florets formation
White cabbage (Brassica oleracea L. var. capitata)	Early ripe F1 Aurora	Head formation	(1) 01.07 (2) 15/07	29/07
	Medium-ripe Slava 1355		(1) 09/07; (2) 22/07	22/08
Red cabbage (Brassica oleracea var. capitata f. rubra)	Kamennaya Golovka 447		(1) 09/07; (2) 22/07	22/08
Savoy cabbage (Brassica oleracea L. var. sabauda)	Vertu 1340
	Melissa F1
Cauliflower (Brassica oleracea var. botrytis)	Freedom F1		(1) 22/07; (2) 02/08	12/08

presents the cultivars used, the phenological phases corresponding to the Se biofortification treatments and the related dates, and the harvest dates.

The plants were sprayed with a Solo handheld backpack sprayer at a working solution rate of 300 L ha⁻¹, which was prepared immediately prior to its use. Selenate solution spraying (50 mg L⁻¹) was carried out in the evening to avoid burning cabbage leaves and the quick evaporation of the solution from the leaf surface. The total amount of sodium selenate per plant was 5 mg per two treatments (26.5 µM). To reduce the negative effect of leaf wax on the efficiency of Se absorption, an adjuvant ‘Atomic’ (OOO ‘Akvalar’, Moscow, Russia) was used in a dose of 10 mL L⁻¹ of sodium selenate solution.

During the growing season, hoeing and manual weeding were carried out. Protection against herbivory was practiced using a 4-fold treatment with insecticides BI-58 New (BASF Societas Europaea, Ludwigshafen, Germany) and Decis Profi (Bayer Crop Science, Monheim, Germany).

2.2. Sample Preparation

After harvesting and removing soil particles, segments of cabbage heads were separated from at least 10 plants and homogenized. The plants were dried at 70 °C to a constant weight and homogenized, and the resulting powders were used for the determination of the mineral composition. To determine the Se content, homogenized plant drying was achieved at room temperature to exclude the Se losses.

2.3. Elemental Composition

The Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Na, Ni, P, Pb, Si, Sn, Sr, V and Zn contents in the dried homogenized plant samples were assessed using ICP-MS on a Nexion 300D quadruple mass-spectrometer (Perkin Elmer Inc., Shelton, CT, USA), equipped with the seven-port FAST valve and ESI SC DX4 autosampler (Elemental Scientific Inc., Omaha, NE, USA) at the Biotic Medicine Center (Moscow, Russia). Rhodium 103 Rh was used as an internal standard to eliminate instability during the measurements. Quantitation was performed using external standards (Merck IV, multi-element standard solution); the Perkin–Elmer standard solutions for P, Si and V, and all the standard curves, were obtained at five different concentrations. For quality control purposes, the internal controls and reference materials were tested together with the samples daily. Microwave digestion of the samples was carried out with sub-boiled HNO₃, diluted at a ratio of 1:150 with distilled deionized water (Fluka No. 02, 650 Sigma-Aldrich, Co., St. Louis, MO, USA) in the Berghof SW-4 DAP-40 microwave system (Berghof Products + Instruments Gmb H, 72, 800 Eningen, Germany). The instrument conditions and acquisition parameters were as follows: plasma power and argon flow, 1500 and 18 L min⁻¹, respectively; aux argon flow, 1.6 L min⁻¹; nebulizer argon flow, 0.98 L min⁻¹; a sample introduction system, ESI ST PFA concentric nebulizer and ESI PFA cyclonic spray chamber (Elemental Scientific Inc., Omaha, NE, USA) were used; the sampler and slimmer cone material was platinum; the injector used was an ESI Quartz 2.0 mm I.D.; sample flow, 637 L min⁻¹; standard internal flow, 84 L min⁻¹; dwell time and acquisition mode, 10–100 ms with peak hopping for all the analytes; sweeps per reading, 1; reading per replicate, 10; replicate number, 3; DRC mode, 0.55 mL min⁻¹ ammonia (294993-Aldrich Sigma-Aldrich, Co., St. Louis, MO 63103, USA) for Ca, K, Na, Fe, Cr, and V, optimized individually for RPa and RPq; and STD mode for the remaining analytes at RPa = 0 and RPq = 0.25 [26].

Trace levels of Hg in the samples were not taken into account, and accordingly, this element was not included in the tables.

2.4. Determination of Selenium

The selenium was analyzed using the fluorimetric method previously described for tissues and biological fluids [27]. Dried homogenized samples were digested via heating with a mixture of nitric and perchloric acids, subsequent reduction of selenate (Se⁺⁶) to selenite (Se⁺⁴) with a solution of 6 N HCl, and the formation of a complex between Se⁺⁴ and 2,3-diaminonaphtalene. The calculation of the Se concentration was achieved by recording the piazoselenol fluorescence value in hexane at 519 nm λ emission and 376 nm λ excitation. Each determination was performed in triplicate. The precision of the results was verified using the Mitsuba reference standard of Se-fortified stem powder in each determination, with a Se concentration of 1865 μg kg⁻¹ (Federal Scientific Vegetable Center).

2.5. Statistical Analysis

Data were processed by analysis of variance, and mean separations were performed through Duncan’s multiple range test, with reference to the 0.05 probability level, using the SPSS software version 27. Data expressed as percentages were subjected to angular transformation before processing.

3. Results and Discussion

No interactions between the research year and Brassica oleracea botanical variety arose, and therefore, the results have been expressed as the means of the two-year data.

B. oleracea plants are known to be relatively tolerant to moderate concentrations of Se. To minimize the effect of the soil–Se interaction, in the present research, a foliar application of sodium selenate was practiced using similar concentrations (50 mg L⁻¹) for all the botanical varieties examined. Previous investigations demonstrated that this concentration did not demonstrate significant effects on plant growth in some cases [10] but was beneficial to kohlrabi [7,28], broccoli [11,29], and cauliflower [30,31,32].

3.1. Mineral Composition

The high K, Ca, P and Fe levels in Brassica oleracea plants of the different botanical varieties examined were consistent with the published data [33,34]. A comparison of the mineral profiles of eight B. oleracea representatives carried out in the present work revealed the highest K levels in kohlrabi, Savoy cabbage and cauliflower; Ca was found in early ripe cabbage and broccoli, and P was observed in kohlrabi and broccoli. Meanwhile, the highest levels of Fe accumulation were recorded in broccoli and red cabbage (

Table 3. Mineral composition of Brassicaceae crops not fortified with Se (mg kg⁻¹ d.w.).

	Early Ripe White Cabbage	Medium- Ripe White Cabbage	Cauliflower	Broccoli	Red Cabbage	Savoy Cabbage *	Savoy Cabbage **	Kohlrabi	M ± SD
Ca	5400 a	2708	3031 bc	4535 a	2998 bc	2031 d	1456 e	3620 b	3222 ± 1283
K	26,411 bc	24,088 c	32,400 ab	29,099 b	27,086 bc	29,896 b	32,605 ab	39,640 a	30,153 ± 4817
Mg	1482 bc	997 bd	1573 b	2201 a	1261 c	1256 c	1342 c	1102 d	1402 ± 373
Na	1997 a	856 b	845 b	558 c	408 d	230 e	441 d	839 b	772 ± 548
P	4117 b	2889 c	4611 b	7746 a	2979 c	4596 b	4802 b	6599 a	4792 ± 1665
Al	6.27 d	5.40 d	3.85 e	3.60 e	15.31 b	4.23 e	5.44 d	10.69 c	6.85 ± 4.09
As	0.03 b	0.018 c	0.01 d	0.02 c	0.04 a	0.01 d	0.01 d	0.01 d	0.0185 ± 0.011
Cd	0.04 b	0.021 c	0.05 a	0.04 b	0.01 d	0.02 c	0.02 c	0.04 b	0.030 ± 0.014
Cr	0.31 c	0.97 a	0.28 c	0.17 d	0.72 b	0.31 c	0.33 c	0.20 d	0.411 ± 0.281
Ni	1.24 d	2.82 b	1.35 d	0.44 e	2.97 b	5.78 a	3.08 b	1.88 c	2.45 ± 1.64
Pb	0.09 e	0.34 a	0.19 c	0.07	0.17 c	0.23 b	0.13 d	0.19 c	0.176 ± 0.085
Sn	0.1 b	0.035 c	0.02 d	0.008 e	0.008 e	0.04 c	0.02 d	0.053 a	0.036 ± 0.030
Sr	22.07 b	13.1 d	12.89 d	37.05 a	11.51 d	6.87 e	3.67 f	15.97 c	15.39 ± 10.36
V	0.05 c	0.12 b	0.03 e	0.04 d	0.20 a	0.04 d	0.03 e	0.04 d	0.071 ± 0.065
B	4.8 c	19.5 a	2.18 e	10.02 b	3.91 d	3.69 d	5.38 c	8.93 b	7.30 ± 5.60
Co	0.13 de	0.18 b	0.09 f	0.09 f	0.17 bc	0.19 b	0.15 cd	0.11 e	0.139 ± 0.040
Cu	2.18 e	3.64 d	3.51 d	5.41 b	2.73 e	4.25 cd	4.28 c	4.38 c	3.80 ± 1.02
Fe	48.72 d	72.9 b	59.44 bc	110 a	118 a	52.16 cd	63.1 b	43.15 d	70.93 ± 28.17
I	0.95 a	0.24 e	0.11 f	0.31 cd	0.34 c	0.40 b	0.27 de	0.23 e	0.356 ± 0.255
Li	0.07 c	0.092 b	0.04 d	0.04 d	0.22 a	0.10 b	0.09 b	0.02 e	0.084 ± 0.062
Mn	16.39 d	24.00 bc	17.96 cd	26.45 b	23.48 bc	34.99 a	21.73 c	9.33 e	21.79 ± 7.58
Mo	0.83 a	0.46 c	0.63 b	0.86 a	1.05 a	0.59 b	1.07 a	0.6 b	0.76 ± 0.23
Se	0.11 c	0.12 bc	0.13 ab	0.09 c	0.07	0.09 c	0.06 d	0.15 a	0.103 ± 0.030
Si	2.57 b	3.63 a	2.14 c	2.63 b	3.67 a	2.62 b	3.58 a	2.7 b	2.94 ± 0.59
Zn	25.9 d	13.1 e	52.55 b	80.3 a	26.84 d	31.73 c	33.95 c	29.64 c	36.75 ± 20.72

* Vertu cv; ** Melissa cv. Along each line, values with the same letters do not differ statistically according to Duncan’s test at p < 0.05. M: means of the values related to the 8 B. oleracea botanical varieties examined for each element; SD: standard deviation.

). Among the heavy metals, Sr prevailed in broccoli, and Cr prevailed in medium-ripe white cabbage of the untreated plants. The same trend was recorded in the Se-biofortified plants (

Table 4. Mineral composition of Brassicaceae crops fortified with Se (mg kg⁻¹ d.w.).

	Early Ripe White Cabbage	Medium- Ripe White Cabbage	Cauliflower	Broccoli	Red Cabbage	Savoy Cabbage *	Savoy Cabbage **	Kohlrabi	M ± SD
Ca	5026 a	4981 a	2597 c	6169 a	2840 c	3128 c	2699 c	3951 b	3924 ± 1333
K	23,729 c	26,651 bc	36,654 a	35,849 a	24,576 bc	25,781 bc	30,792 b	38,889 a	30,365 ± 6034
Mg	1298 c	1089 c	1914 b	2555 a	1117	1281 c	1261 c	1316 c	1479 ± 504
Na	1637 a	775 c	1564 a	470 e	481 e	609 d	336 f	1111 b	873 ± 507
P	3737 b	3133 cd	4582 b	6831 a	2624 d	3905 b	4113 b	6086 a	4376 ± 1430
Al	5.53 c	14.6 b	4.28 d	3.1 e	3.99 d	18.75 a	3.74 d	13.53 b	8.44 ± 6.17
As	0.04 a	0.0088 c	0.01 b	0.01 b	0.01 b	0.04 a	0.01 b	0.01 b	0.017 ± 0.014
Cd	0.07 a	0.024 d	0.04 b	0.04 b	0.007 e	0.03 c	0.02 d	0.03 c	0.033 ± 0.019
Cr	0.74 a	0.25 c	0.38 b	0.17 d	0.38 b	0.36 b	0.12 e	0.21 c	0.326 ± 0.194
Ni	1.11 d	2.4 c	1.28 d	0.54 f	2.90 b	4.13 a	2.94 b	0.87 e	2.02 ± 1.26
Pb	0.13 d	0.24 b	0.21 b	0.09 e	0.12 d	0.50 a	0.19 c	0.09 e	0.196 ± 0.135
Sn	0.06 b	0.021 d	0.006	0.02 d	0.04 c	0.01 e	0.01 e	0.042 a	0.026 ± 0.019
Sr	19.54 c	25.4 b	10.18 e	46.49 a	10.34 e	13.14 d	7.01 f	16.46 c	18.57 ± 12.72
V	0.04 c	0.019 e	0.04 c	0.03 d	0.04 c	0.19 a	0.03 d	0.07 b	0.057 ± 0.056
B	3.5 de	21.9 a	1.76 f	11.19 b	3.12 e	4.09 d	3.9 d	7.5 c	7.12 ± 6.68
Co	0.10 de	0.15 c	0.11 d	0.06 f	0.19 b	0.19 b	0.27 a	0.08 e	0.144 ± 0.070
Cu	1.98 e	3.54 bc	3.53 bc	4.69 a	2.82 d	2.94 d	3.24 c	4.13 ab	3.36 ± 0.83
Fe	48.01 de	40.2 e	65.9 c	98.19 b	49.24 d	121 a	40.14 e	60.25 c	65.37 ± 29.35
I	0.97 a	0.32 c	0.48 b	0.23 e	0.25 de	0.29 cd	0.45 b	0.08 f	0.384 ± 0.268
Li	0.05 d	0.054 d	0.05 d	0.04 e	0.26 a	0.17 b	0.11 c	0.03 f	0.096 ± 0.081
Mn	16.34 f	17.5 ef	21.75 d	28.21 c	18.59 de	37.94 b	46.39 a	9.09 g	24.48 ± 12.36
Mo	0.62 b	0.37 c	0.66 b	0.61 b	0.58 b	0.64 b	0.86 a	0.55 b	0.611 ± 0.135
Se	4.29 d	1.45 g	17.91 a	7.56 c	3.0 e	2.44 f	3.31 e	13.05 b	6.62 ± 5.89
Si	2.62 cd	3.83 a	2.10 c	2.37 c	3.21 ab	2.89 bc	3.19 b	2.22 d	2.80 ± 0.59
Zn	48.2 b	11.4 f	35.28 c	74.72 a	22.92 e	21.42 e	25.14 de	28.16 d	33.40 ± 19.87

* cv. Vertu; ** cv. Melissa. Along each line, values with the same letters do not differ statistically according to Duncan’s test at p < 0.05. M: means of the values related to the 8 B. oleracea botanical varieties examined for each element; SD: standard deviation.

).

The comparison between the total mineral contents of the eight Brassica oleracea botanical varieties studied highlighted kohlrabi, broccoli and cauliflower as the richest sources of minerals (Figure 1). Selenium supplementation had no significant effect on the total mineral levels in plants, though a tendency of this parameter increase can be noted in the broccoli, cauliflower and medium-ripe white cabbage that was treated with sodium selenate.

Both the control and Se-fortified plants showed the lowest differences between the B. oleracea botanical varieties in terms of macro-elements, which are the most effective for plant growth, and of Si, Cu and Mo (Figure 2). Unlike the latter, the most remarkable variations were recorded for Al, As and heavy metals, especially V. Interestingly, upon receiving the Se supply, the varietal differences in element accumulation decreased for Cr, Ca, Sn, Ni and Mo, while a pronounced increase in the CV values arose for most of the other elements, such as Al, As, Cd, Pb, V, B, Co, Fe, Li and Mn, suggesting the important role of genetic peculiarities in the mineral accumulation changes occurring in the B. oleracea receiving the Se supply (Figure 2).

In this respect, it is important to highlight the different Se effects on the mineral composition of the six Brassica oleracea botanical varieties compared (

Table 5. Significant changes (p < 0.05) (ratio between elements in Se-fortified/control plants) in the elemental composition of Brassica oleracea crops under foliar application of sodium selenate.

	Early Ripe White Cabbage	Medium-Ripe White Cabbage	Cauliflower	Broccoli	Red Cabbage	Savoy Cabbage *	Savoy Cabbage **	Kohlrabi
Macro-elements and Si
Mg	Relatively high stability levels
P
Si
Ca		1.84		1.36		1.54	1.85
K				1.23
Na			1.85			2.65	0.76	1.32
Micro-elements
B	0.73		0.81		0.8		0.72
Co	0.77	0.83	1.22	0.67			1.80	0.73
Cu						0.69	0.76
Fe					0.42	2.32	0.64	1.40
I		1.33	4.36	0.74	0.74	0.73	1.67	0.06
Li	0.71	0.59				1.70		1.50
Mn		0.73			0.79		2.13
Mo	0.75	0.80		0.71	0.55
Zn	1.86		0.67			0.68	0.74
As, heavy metals and Al
Al		2.70			0.26	4.43	0.69	1.27
As	1.33	0.49		0.50	0.25	4.00
Cd	1.75				0.70	1.50		0.75
Cr	2.39	0.26	1.36		0.53		0.36
Pb	1.44	0.71			0.71	2.17	1.46	0.47
Ni						0.71		0.46
Sr		1.94		1.25		1.91	1.91
V	0.80	0.16	1.33	0.75	0.20	4.75		1.75

* cv. Vertu; ** cv. Melissa.

).

3.2. Changes in Elemental Composition under Se Supply

In the present research, a special group of elements showed high concentration stability, such as Mg, P and Si (Table 5). Mg and P are essential elements for plant growth and development, whereas Si is only beneficial, and its accumulation levels, mostly depending on plant genetic peculiarities [35], affect crop productivity, photosynthesis processes and metabolic function regulation. Brassica oleracea plants are not Si accumulators, and usually, their content of this element is within 2–4 mg kg⁻¹ d.w. [36]. Silicon varietal differences in the present work ranged between 2.1 to 3.8 mg kg⁻¹ d.w., similar to previous reports for cauliflower [36].

Though the Brassica oleracea representatives demonstrated a high response variability to the Se supply, it is worth highlighting the antagonistic effect of Se recorded just on four elements: B (early ripe white cabbage, cauliflower, red cabbage and Savoy cabbage, cv. Melissa); Cu in Savoy cabbage; Mo (broccoli, white and red cabbage) and Ni (Savoy cabbage, cv. Vertu and kohlrabi). Contrary to this, only the positive effect of Se was recorded on Ca and Sr in medium-ripe white cabbage, broccoli and Savoy cabbage. Furthermore, red cabbage exclusively demonstrated a decrease of nine element contents, including not only most of the heavy metals as well as As and Al, but also essential micronutrients, such as Mo, Mn and Fe. The antagonistic relationship between Mo and Se was previously reported in Chinese cabbage [13]; however, in this research, a Mo decrease under the Se supply was recorded but only in broccoli and white and red cabbage (Table 5).

In hydroponics, Se utilization was shown to stimulate the accumulation of Fe, Mn, Zn and Cu in Zea mays [37]. Contrarily, the present results related to the foliar selenate application revealed great differences between various Brassica oleracea representatives regarding the changes in these elements’ concentrations, with a statistically significant decrease of Zn and Cu upon Se supply only in some species.

Furthermore, when heavy metal uptake is not detectable, foliar Se supplementation resulted in both a decrease and increase of toxic element concentrations, though in a safe concentration range. Indeed, the Se supply increased the Cr levels in early ripe white cabbage, cauliflower and Savoy cabbage (cv. Melissa), while a negative effect was recorded in medium-ripe white cabbage and red cabbage. An increase in the V content was detected in medium-ripe white cabbage, kohlrabi and cv. Vertu of Savoy cabbage, but was decreased in red cabbage. Based on this study’s evidence, the interactions of Se with heavy metals are complex. Indeed, the well-known Se protective effect of plants against heavy metals was accompanied by heavy metals increasing in several B. oleracea representatives upon Se supply: Al and V in medium-ripe white cabbage, Savoy cabbage cv. Vertu and kohlrabi, and Pb in early ripe white cabbage and Savoy cabbage. An analogous phenomenon was previously reported for Brassica juncea and Brassica napus [38], despite the intensive growth stimulation due to Se treatment. The same ability has been detected in many mushroom species under Se and heavy metals anthropogenic uptake [39].

The reaction of Brassica oleracea plants to Se treatment in terms of the Ca and Sr concentration changes is consistent with the literature reports and is connected to the close chemical structure of Sr and Ca [40]. Moreover, it is worth mentioning the great species differences regarding their Sr/Ca changes due to receiving a supply of Se. Indeed, no effects on the accumulation of these elements were recorded in early ripe white cabbage, cauliflower, red cabbage and kohlrabi, while a significant increase in the Sr/Ca content arose in medium-ripe white cabbage, broccoli and Savoy cabbage (Table 5).

Overall, the most intensive changes of elemental composition were recorded in Savoy cabbage, including 13–14 elements (Table 5). Contrary to this, floret cabbages (broccoli and cauliflower) showed the lowest significant mineral changes (only 7–8 out of 25 elements studied). In general, the intensity of the mineral composition changes decreased as follows: Savoy cabbage (13–14) > medium-ripe white cabbage (12) > red cabbage (11) and kohlrabi (10) > early ripe white cabbage (10) > broccoli (8) > cauliflower (7).

3.3. Correlations

The complexity of the Se effects on the mineral contents in Brassica oleracea plants entails the need to also study its implications on the element relationships in the control and Se-fortified species. For this purpose, separate calculations of correlation coefficients between elements were completed for the control plants and plants biofortified with Se (

Table 6. Correlations between the analyzed elements in Brassicaceae plants.

Elements

Ca

K

Mg

Na

P

Al

As

Cd

Cr

Ni

Pb

Sn

Ca

1

−0.148

0.475

0.746 g

0.318

0.028

0.454

0.585

−0.272

−0.723 g

−0.509

0.523

K

0.015

1

−0.066

−0.196

0.593

0.111

−0.596

0.392

−0.651 k

−0.079

−0.176

−0.032

Mg

0.434

0.514

1

0.025

0.641

−0.416

0.073

0.484

−0.558

−0.575

−0.737 g

−0.256

Na

0.046

−0.155

0.022

1

−0.117

−0.082

0.281

0.551

−0.074

−0.530

−0.241

0.837 c

P

0.439

−0.093

0.761 f

0.029

1

−0.289

−0.409

0.565

−0.794 e

−0.430

−0.547

−0.430

Al

−0.011

−0.493

−0.444

−0.036

−0.112

1

0.615

−0.437

0.323

0.055

0.017

−0.064

As

0.061

−0.075

−0.221

0.305

−0.227

0.355

1

−0.344

0.419

−0.196

−0,248

0.078

Cd

0.486

0.072

0.317

0.735 g

0.277

−0.124

0.580

1

−0.634

−0.700 1

−0.350

0.301

Cr

0.065

−0.033

−0.222

0.691 j

−0.410

−0.069

0.710 1

0.666 k

1

0.263

0.699 d

−0.159

Ni

−0.573

0.089

−0.596

−0.518

−0.668 k

0.435

0.288

−0.552

−0.088

1

0.511

−0.071

Pb

−0.348

0.170

−0.268

−0.156

−0.341

0.676 k

0.536

−0.115

0.035

0.769 f

1

−0.086

Sn

0.351

−0.582

−0.301

0.384

−0.668 k

−0.103

0.287

0.387

0.611

−0.376

−0.532

1

Sr

0.922 b

0.211

0.679k

−0.146

0.582

−0.092

−0.114

0.301

−0.169

−0.538

−0.298

0.071

V

−0.291

−0.215

−0.178

−0.106

−0.027

0.696 h

0.644

−0.033

0.090

0.570

0.812 e

−0.216

B

0.619

−0.035

−0.016

−0.226

0.041

0.367

−0.332

−0.141

−0.357

−0.110

−0.040

−0.071

Co

−0.655 k

0.171

−0.576

−0.550

−0.624

0.035

0.010

−0.581

−0.230

0.840 c

0.425

−0.369

Cu

0.325

0.024

0.638

−0.313

0.778 f

−0.004

−0.667 h

−0.257

−0.790 e

−0.428

−0.265

−0.411

Fe

0.184

0.112

0.472

−0.078

0.394

0.392

0.417

0.158

0.013

0.086

0.506

−0.262

I

0.094

0.281

−0.109

0.579

−0.339

−0.337

0.565

0.737 g

0.789 e

−0.130

−0.041

0.359

Li

−0.549

0.054

−0.426

−0.511

−0.621

−0.001

0.116

−0.618

0.059

0.744 g

0.306

−0.024

Mn

−0.312

0.535

0.097

−0.583

−0.046

−0.083

0.139

−0.223

−0.366

0.572

0.500

−0.665 k

Mo

−0.482

0.301

0.155

−0.164

0.127

−0.471

0.107

0.029

−0.109

0.167

0.045

−0.293

Se

−0.171

−0.049

0.489

0.572

0.555

−0.219

−0.328

0.230

−0.064

−0.616

−0.301

−0.142

Si

−0.028

0.082

−0.622

−0.516

−0.715 g

0.251

−0.072

−0.497

−0.139

0.632

0.267

−0.075

Zn

0.616

0.333

0.856 b

0.121

0.689 d

−0.550

0.057

0.573

0.103

−0.688 h

−0.456

0.158

Sr

V

B

Co

Cu

Fe

I

Li

Mn

Mo

Se

Si

Zn

Ca

0.824 c

−0.066

0.038

−0.544

−0.24

0.074

0.609

−0.304

−0.396

−0.005

0.371

−0.400

0.308

K

−0.139

−0.519

−0.301

−0.469

0.424

−0.440

−0.395

−0.507

−0.522

−0.058

0.378

−0.476

0.497

Mg

0.764 f

−0.349

−0.22

−0.661 k

0.397

0.430

0.082

−0.307

0.167

0.336

−0.214

−0.458

0.952 a

Na

0.331

−0.158

0.044

−0.296

−0.600

−0.393

0.729 g

−0.314

−0.560

−0.112

0.437

−0.198

−0.205

P

0.612

−0.633

−0.043

−0.703 h

0.768 f

−0.01−

−0.161

−0.681 k

−0.161

0.024

0.186

−0.579

0.925 b

Al

−0.185

0.736 g

−0.125

0.223

−0.408

0.334

0.013

0.633

−0.033

0.357

−0.082

0.462

−0.389

As

0.265

0.772 f

−0.061

0.193

−0.618

0.619

0.498

0.700 h

0.012

0.485

−0.336

0.527

−0.219

Cd

0.531

−0.663 k

−0.128

−0.884 b

0.134

−0.373

0.043

−0.837 c

−0.528

−0.316

0.671 j

−0.834 c

0.590

Cr

−0.311

0.798 e

0.563

0.659 k

−0.406

0.331

−0.114

0.618

0.200

−0.149

−0.115

0.731 g

−0.707 h

Ni

−0.738 g

0.153

−0.127

0.847 b

−0.009

−0.202

−0.083

0.449

0.620

−0.147

−0.341

0.246

−0.533

Pb

−0.513

0.319

0.536

0.575

−0.074

−0.202

−0.421

0.147

0.179

−0.705 h

0.332

0.242

−0.606

Sn

0.061

−0.270

−0.023

0.024

−0.508

0.679 k

0.796 e

−0.294

−0.385

−0.258

0.418

−0.182

−0.428

Sr

1

−0.135

0.232

−0.623

0.273

0.380

0.242

−0.383

−0.104

0.016

0.194

−0.340

0.672 k

V

−0.239

1

0.202

0.509

−0.467

0.641

−0.024

0.860 b

0.134

0.220

−0.276

0.670 k

−0.463

B

0.574

−0.293

1

0.181

0.251

0.111

−0.191

−0.163

−0.003

−0.466

0.279

0.389

−0.207

Co

−0.643

0.164

−0.203

1

−0.277

0.019

0.122

0.677 k

0.561

−0.049

−0.420

0.646

−0.805 e

Cu

0.570

−0.172

0.420

−0.404

1

0.116

−0.564

−0.473

0.257

−0.123

−0.031

−0.392

0.665 k

Fe

0.376

0.727 g

−0.091

−0.359

0.265

1

−0.195

0.567

0.353

0.497

−0.533

0.367

0.360

I

−0.146

−0.201

−0.296

0.006

−0.731 g

−0.296

1

0.087

−0.045

0.212

−0.112

0.330

−0.266

Li

−0.455

0.332

−0.365

0.628

−0.422

0.063

−0.184

1

0.393

0.476

−0.642

0.674 j

−0.513

Mn

−0.158

0.301

−0.238

0.691 1,j

−0.035

0.131

0.021

0.276

1

0.012

−0.600

0.096

0.065

Mo

−0.393

0.096

−0.735 g

0.479

−0.154

−0.095

0.244

0.157

0.721 g

1

−0.779 f

0.415

0.180

Se

−0.055

−0.132

−0.306

−0.546

0.424

0.112

−0.126

−0.484

−0.350

0.099

1

−0.524

−0.015

Si

−0.122

−0.112

0.525

0.634

−0.295

−0.398

−0.030

0.415

0.213

−0.289

−0.826 d

1

−0.663 k

Zn

0.712 h

−0.236

−0.127

−0.621

0.334

0.345

0.200

−0.402

−0.010

0.198

0.269

−0.609

1

Dark segments correspond to control plant data; white segments correspond to Se-treated plants. Significance: p <: ^a 0.0001; ^b 0.001; ^c 0.002; ^d 0.003; ^e 0.005; ^f 0.01; ^g 0.02; ^h 0.03, ^j 0.04; ^k 0.05. Correlations with r values < 0.644 are not statistically significant at p > 0.05. ‘¹’ is usually used in the correlation Tables and it indicates that the correlation coefficients between the same elements (K–K, Ca–Ca, Na–Na, etc.) are equal to 1.

). Data presented in Table 6 prove that a Se supply may not only affect a plant’s mineral composition but also change the relationships between the elements. In general, a significantly higher frequency of element relationships was recorded in the control plants (42 at p < 0.05) than in the Se-fortified ones (32 at p < 0.05).

3.3.1. Stable Interactions

Three stable, highly significant correlations between Sr and Ca (r = 0.824–0.922), Co and Ni (r = 0.847–0.840), and Zn and Mg (r = 0.856–0.952) in the control and Se-fortified plants, were recorded. Other statistically significant correlations, with lower coefficient values, are as follows: Sr–Mg (0.764–0.679); Cr–Cd (0.634–0.666); V–Al (0.736–0.696), Cu–P (0.768–0.778); Cu–As (0.618–0.667); Li–Co (0.677–0.628).

The stability of the Sr–Ca relationship is undoubtedly connected by the similarity of the Ca and Sr atoms, resulting in the fact that Sr constantly accompanies Ca in the environment [40].

The close relationship between Co and Ni is presumably connected by their similar ABC transporters, providing their synchronous movement in plants [41,42]. Nevertheless, the present results are the first ones to record the lack of a significant Se effect on the Co–Ni relationship. Co is considered a beneficial element for plant growth, participating in Fe, Ni and Zn interactions to maintain cellular homeostasis [43]. The importance of Ni for a plant’s growth and development is connected with the ability of Ni-containing enzymes to catalyze the conversion of urea into ammonium ion, used by plants as a source of N; at present, this element is considered essential for plants [44]. Furthermore, Ni is supposed to play an important role in plant antioxidant metabolism [45].

The high Zn–Mg relationship was previously reported by Prasad et al. [46]. Both elements play an important role in cell division and cell lengthening, which may also be responsible for a plant’s height increase [47]. Zinc participates in gene expression, protein synthesis, carbohydrate metabolism, photosynthesis and the defense against plant diseases in most plant species [38]. The essential role of Mg is connected with its involvement in photosynthesis, net assimilation and relative growth and yield [46]. Carbohydrate, protein and chlorophyll formation are significantly reduced in magnesium- and zinc-deficient plants [48].

Taking into account the mentioned stability of the element correlations, the relationships Mg/Zn, Ni/Co and Sr/Ca represent important characteristics of Brassica oleracea plants. In this respect, among the eight representatives of Brassica oleracea plants examined, six demonstrated a Ca/Sr ratio close to 244; broccoli was characterized by the lowest Ca/Sr ratio (122–133), while cv. Vertu of Savoy cabbage showed the highest Ca/Sr ratio of 385–397.

3.3.2. Differences in Se Accumulation

The phenomenon of species and varietal differences in Se biofortification levels of agricultural crops has been poorly understood thus far. Plant classifications in Se hyperaccumulators, Se indicators and Se non-accumulators are highly connected with the metabolism of S, which is a chemical analog of Se [49]. Nevertheless, this statement does not explain the significant differences in Se biofortification levels, particularly between Brassica oleracea crops. Indeed, the results of the present investigation revealed great differences in this parameter, ranging from 12 in medium-ripe white cabbage to 138 in cauliflower, in the same growth conditions and foliar selenate concentration supply (Figure 3).

The results indicated a decrease of the Se biofortification value according to the following sequence: cauliflower > kohlrabi > broccoli > Savoy cabbage, cv. Melissa > red cabbage > early ripe white cabbage > Savoy cabbage, cv. Vertu > medium-ripe white cabbage. From the correlations between the elements examined in the eight different Brassica oleracea representatives, a high negative correlation coefficient arose between the Se biofortification values and Si contents (Figure 4). Recent examples of the antagonistic relationship between these two micro-elements were reported only for selenite under Si supply [50].

Taking into account the relative stability of the Si levels under Se supply (Table 5), it may be assumed that the differences in the ability of Brassica oleracea plants to accumulate Se depend on the initial content of Si. The previously recorded increase of Se content in the chervil, fortified with Se under Si inorganic forms and nano Si supply [21,51,52], may be attributed to Si-micro-dose growth stimulation, which did not affect plant Si concentrations. However, the possible connection of this phenomenon with the well-known protective effect of Si against environmental stresses [53] needs further investigation.

Selenium and Si are known to improve plant tolerance to Cd stress [18]. In this respect, a previous investigation of the Se–Si–Cd relationship in Chinese cabbage revealed the importance of Si/Se antioxidant defense improvement for alleviating Cd stress, with a significantly more powerful effect of Se compared to Si [54]. It may be assumed that a significant decrease of the Cd–Si correlation coefficient, from −0.834 to −0.497 under Se supply, may be attributed to the more intensive protective participation of Se in Se-enriched plants, though further investigations are needed to confirm this hypothesis.

3.3.3. Significant Changes in Element Correlations

In the present research, the correlations between the mineral elements in eight Brassica oleracea botanical varieties were associated with high correlation coefficients between Zn and P (+0.925), Zn and Co (−0.850), Cd and Co (−0.884), Cd and Li (−0.837), V and Li (+0.860) and Na and Sn (+0.837) in the control plants (Table 6). Sodium selenate supplementation reduced these values, leading to non-significant correlations between these elements in Se-treated plants (Table 6).

Contrarily, the Se-fortified plants demonstrated a higher V–Pb correlation coefficient compared to that of the control plants (r = 0.319 and 0.812, respectively), suggesting the existence of a Se-stimulation effect on the V/Pb relationship.

Contrary to the literature reports [46], the correlation between Zn and P had a high correlation coefficient, similar to the Zn–Mg correlation, which is consistent with the literature data [46].

The effects of the P amendments on a plant’s Zn content depend on the soil’s Zn concentration and type [55]. In this respect, Pongrac et al. [56] were unable to reveal a negative effect of P on Zn accumulation in red cabbage, contrary to other observations [57], which may relate to the precipitation of insoluble Zn₃(PO₄)₂ reducing Zn uptake by roots.

The close relationship between the values of Zn⁺² and the Co⁺² atomic radius (0.74 Å and 0.76 Å, respectively) creates the basis of a possible Zn substitution by Co, which has been recorded in algae and plant enzymes [44]. Indeed, Co may inhibit the accumulation of Zn [58], as witnessed by the negative correlation between these two elements in control plants. In this respect, the imbalance of this relationship, caused by the Se supply, may be beneficial for plant growth and development, though further investigations are necessary to prove this hypothesis.

The phenomenon of the V–Pb interaction increase due to the Se supply may become of special importance, as at low concentrations, V is known to reduce the detrimental effects of Pb [59,60].

3.3.4. Cromium Relationships

The number of changes in the correlation coefficients due to the Se supply was the lowest for K, Al and B (only K-Cr, Al-Pb and B-Mo) (

Table 7. Changes in mineral correlations of Brassicaceae plants under Se biofortification.

	Ca	K	Mg	Na	P	Al	As	Cd	Cr	Ni	Pb	Sn	Sr	V	B	Co	Cu	Li	Mn	Mo	Se	Si
Na	−
P			+
Cd				+
Cr		−		+	−		+
Ni	−				+			−
Pb			−			+			−	+
Sn				−	+
Sr	+									−
V							−	−	−		+
Co	+		−		−			−	−
Cu									+
Fe												−		+
I				−				+	+			−					+
Li					−		−	−		+				−
Mn												+				+
Mo											−				+				+
Se								−												−
Si					+			−	−				−	−				−			+
Zn					−				−	+			+			−	−					−

significant ‘r’ decrease (p < 0.05); significant ‘r’ increase (p < 0.05)

). Contrary, among the 25 elements studied, Cr showed the highest number of changes in the relationship with other elements, such as macro- (K, Na and P), micro- (Si, Zn, Cu, Co and I), and toxic elements (As, Pb and V) (Figure 5). The protective role of Se against heavy metals, including Cr, is one of the crucial Se beneficial effects and is explained by the enhancement of a plant’s antioxidant status, inhibition of heavy metals absorption and nutrition improvement [18]. In hydroponics, an increase of Na, Cu and Zn contents in B. campestris sp. Pekinensis, grown under Cr stress, was recorded under Se supply [61], and an analogous effect was reported for B. napus [62]. Cr toxicity in Indian mustard revealed a protective effect of Se, reflected in the increase of both antioxidant activity and Ca and Mg accumulation, but decreased Cr content [63]. Nevertheless, no attempts have been made to assess the correlation coefficients of Cr with other elements in ordinary growing conditions and under Se treatment.

The broad relationship of Cr with other elements relates to the element’s effect on photosynthesis, chlorophyll accumulation and plant defense systems [64]. Lacking Cr–Zn, Cr–V and Cr–Si correlations and raising a strong Cr–I interaction due to the Se supply may greatly change the mechanism of plant antioxidant protection. Indeed, I, Si and Zn are important components in plant antioxidant defense. The protective role of I against Cr toxicity was previously described for chervil plants [21]. Silicon application stimulated the production of antioxidant enzymes in conditions of Cr stress [65] and caused predominant Cr accumulation in roots, decreasing its levels in leaves and eliciting co-precipitation [66]. The protective effect of Zn against Cr toxicity was recorded previously in rice via stimulation of antioxidant enzymes biosynthesis [67,68]. Among the above elements, only iodine and Na demonstrated a significant increase in the correlation coefficient with Cr under Se supply, which indirectly suggests changes in the participation intensity of the different elements to the antioxidant defense of Brassicaceae plants under Se supply.

Furthermore, an imbalance of the synergic Cu–Cr, [69] Pb–Cr, V–Cr and K–Cr interactions due to the Se supply may be beneficial for Se-fortified plants. Investigations of the V and Cr contents in different medicinal plants demonstrated that the accumulation levels depend on the plant species and are not a function of the metal species concentration in soil [70]. The participation of Cr, V and P in the soil’s bacterial community structure [71] also indirectly indicates the possible effect of Se supply on soil characteristics changes.

Cr may reduce the accumulation of P [64], and according to the results of the present research, Se supplementation decreases the Cr influence. Taking into account the contradictory data related to the Cr–P, Cr–Cu and Cr–Zn interactions, Sharma et al. suggested that Cr may disturb the mineral balance in plants [64]. In the present experiment, the Se application decreased the Cr correlations with the K, P, Si, Zn, Co, Pb, and V contents and significantly improved those with I and Na.

4. Conclusions

The recorded variations in the element correlations, as a result of the sodium selenate foliar supply, allow for highlighting important peculiarities of the Se effects on Brassica oleracea plants, such as the stability of the Ca–Sr, Co–Ni and Zn–Mg interactions, a significant decrease of correlation coefficients for Zn with P and Co; Cd with Co, Si and Li; Cr with V, Cu, Si, Zn and P; Pb with Mg; and a remarkable increase of the correlations of Se with Si; Pb with V and Ni; and Cr with I and Cu. The greatest changes in mineral status, in response to the foliar Se treatment, were recorded in Savoy cabbage and medium-ripe white cabbage, and the lowest in floret cabbage (cauliflower and broccoli). The negative correlation between Si and the Se biofortification level may be considered the first explanation of the differences between B. oleracea botanical varieties in Se accumulation intensity. Overall, the present research results provide a deep outlook of the complex relationships between the mineral elements in Brassica oleracea plants, which may serve as an important reference for further investigations regarding biofortification processes with essential elements.