Effects of Selenium/Iodine Foliar Application and Seasonal Conditions on Yield and Quality of Perennial Wall Rocket

Abstract

The biofortification of leafy vegetables with selenium (Se) and iodine (I) provides the basis for the Se/I status optimization and preservation of human health. The effect of foliar Se, I, and Se + I supply in three different crop cycles (autumn, autumn–winter, and winter) on yield, quality, and mineral composition of wall rocket leaves was investigated using biochemical and ICP-MS methods of analysis. Joint foliar supply with selenate/iodide increased yield, antioxidant activity, total phenolic, ascorbic acid, and protein levels by 1.63, 1.24, 1.22, 1.25, and 1.50 times, respectively, and the content of Ca, Mg, P, K, Fe, Cu, and Zn by 1.27, 1.24, 1.35, 1.46, 3.67, 2.76, and 1.44 times, respectively. High correlations between Se, antioxidants, P, Mg, and Ca (r > 0.80) as well as between yield and K/protein content were recorded. Despite a significant decrease in yield, protein, and K, Fe, Cu, and Mn contents in the third crop cycle, compared to the first one, 50 g of wall rocket biofortified with Se/I may provide up to 100% of the Se adequate consumption level, 34.3% of I, 9% of K, 24% of Fe, and 17.7% Ca. The results of the present research confirm the high efficiency of Se/I supply to produce D. tenuifolia leaves as a new functional food.

1. Introduction

Selenium and iodine are essential microelements for human organism, responsible for antioxidant defence, high immunity, and brain activity, playing an important role in growth and development, protection against chronic diseases including cancer, and heart failure [1]. Selenium and iodine deficiency is widespread in many countries in the world, decreasing life expectation, human education, and labour efficiency, thus causing significant economic losses [2]. The metabolism of these elements is closely related to each other, as Se is important to an active centre of tri-iodothyronine deiodinases participating in the thyroid hormone biosynthesis [3]. In this respect, the optimization of the human Se and I status should be carried out simultaneously [4]. Among different approaches for solving this problem (food supplements and iodized salt), the joint biofortification of agricultural crops with Se and I has become more and more attractive due to the so-called buffer effect of plants preventing Se/I toxicosis in humans and thanks to the low cost of the applied technology [5,6].

Many studies have been carried out regarding the joint Se/I biofortification of agricultural crops, i.e., in carrot and lettuce [7,8,9,10], chicory [11], potato [12], Indian mustard [13], chickpea [14], chervil [15], pumpkin seedlings [16], kohlrabi [17], radish [18], buckwheat [19], spinach [20], and pea [21]. The results demonstrated the high variability of yield, antioxidant status, and the mineral composition of biofortified plants, which greatly hampered the development of industrial technology. The reason of the latter situation is connected with several factors including (1) the extremely narrow concentration ranges suitable for safe biofortification with Se and I [1], (2) species and varietal differences in plant tolerance to Se and I [22], (3) a different efficiency of Se and I biofortification which hinders the production of Se/I-enriched plants, thereby allowing them to sufficiently meet the adequate Se/I consumption levels (70 and 150 µg day⁻¹, respectively) [1,22], and (4) the unpredictable Se/I interaction during biofortification, which may result either in synergism or antagonism between the two microelements, or no effects [11,19].

To date, Se and I are essential for mammals, while they are considered only beneficial in plants, showing growth stimulation and stress protection at low concentrations, which opens new opportunities for the yield improvement of agricultural crops [1]. However, iodine essentiality was recorded via plant protein iodination [23], whereas that of Se was previously demonstrated in algae [24]. The investigation by Rakoczy-Lelek et al. [25] demonstrated that the biofortification of plants with iodide resulted in the formation of iodotyrosine, iodobenzoates, iodosalicylates, and plant-derived thyroid hormone analogues.

Among the agricultural crops tested, leafy vegetables are very interesting targets for biofortification due to the possibility of Se/I losses during drying and food processing because of the formation of volatile methyl iodides and methyl selenides [26,27,28]. In addition, fresh leafy vegetables provide the highest levels of other antioxidants (vitamins, polyphenols, sterols, etc.) known to act synergistically with Se in humans [29,30].

The agronomic biofortification of the Brassicaceae species, which are important dietary sources of Ca, Mg, and Fe [14], is highly desirable due to high levels of sulphur, with the latter being a Se chemical analogue easily substituted with Se in living organisms [31]. Furthermore, selenium is known to form Se derivatives of glucosinolates present in Brassicaceae, with higher anti-carcinogenic activity compared to the unsubstituted compounds [32].

Among the leafy Brassicaceae species, only Indian mustard has been jointly fortified with Se and I [13], and, in this respect, perennial wall rocket (Diplotaxis tenuifolia L.-DC) may become an attractive target of Se/I biofortification. Indeed, Diplotaxis tenuifolia is a perennial plant mostly cultivated to be commercialized as baby leaves rich in glucosinolates, vitamin C, and polyphenol content [33,34,35,36]. The mentioned plant is highly valuable as a food and a spice and is popular in medicine, providing protection against cancer and heart failure due to its high antioxidant activity [37,38]. Its remarkable adaptability and tolerance to salinity, pathogen attack, and diseases [38,39,40,41] as well as quick growth provide additional benefits for wall rocket utilization as a target of Se/I biofortification. To date, no research regarding the joint biofortification of perennial wall rocket with Se and I has been carried out. In previous studies, Se biofortification in hydroponic conditions stimulated wall rocket growth and the accumulation of chlorophyll, polyphenols, and Se [42,43,44], while iodine application via foliar/soil supply did not lead to a significant effect on plant yield but only resulted in tendential chlorophyll accumulation under the foliar treatment [45]. Plant foliar biofortification both with Se [46] and iodine [47] is more efficient than the hydroponic and soil applications, does not cause Se/I interactions with soil components, is easy to perform and, therefore, is preferable to produce functional vegetables enriched with the mentioned essential microelements.

Based on the concepts expressed above, the present work aimed to assess the interactions between foliar joint Se/I biofortification and crop cycle on yield, selenium, iodine, and other antioxidant contents, as well as the mineral composition of D. tenuifolia leaves.

2. Materials and Methods

2.1. Experimental Protocol and Growing Conditions

Research was carried out on Diplotaxis tenuifolia (L.) D.C. cultivar Marte at the Department of Agricultural Sciences, University of Naples Federico II, Portici (Naples), Southern Italy, in 2022–2024 under an unheated greenhouse covered with a thermal polyethylene film in sandy loam soil (76% sand, 17% silt, 7% clay), with 0.12% N, 95.5 ppm NO₃-N, 2.4 ppm NH₄-N, 96.1 ppm P₂O₅, 1822 ppm K₂O, pH 7.2, 2.86% organic matter, and 689 µS cm⁻¹ electrical conductivity. During the cropping seasons, the mean monthly temperatures inside the greenhouse were 18 °C in November, 15 °C in December, 11 °C in January, 14 °C in February, and 18 °C in March.

The experimental protocol was based on the factorial combination of 3 crop cycles (autumn, starting on 2 November; autumn–winter, starting on 9 December; and winter, starting on 24 January) and 3 biofortification treatments (selenium, iodine, and selenium + iodine) plus an untreated control. A randomized complete block design was used for the treatment distribution in the field with three replicates, and each experimental unit had a 1 m² (1 × 1 m) surface area.

Sowing was performed on 2 November, both in 2022 and 2023, with a spacing of 2 cm along the rows which were 5 cm apart. Foliar spraying with selenium, iodine, and selenium + iodine solutions was performed 3 times at 5-day intervals, starting when the leaves were 1 cm long on average, applying the same following doses in each of the three crop cycles: sodium selenate at 50 mg Se L⁻¹ (0.26 mM), potassium iodide at 100 mg I L⁻¹ (0.59 mM), and sodium selenate + potassium iodide at the same mentioned doses. The Se and I doses were chosen based on the tolerable dose of Se in most agricultural crops and the significantly lower ability of plants to accumulate iodine compared to selenium [1,15]. Each solution was applied until runoff at the average volume of 300 mL per plant in each crop cycle, corresponding to the supply of 10 mg of Na and 28 mg of K per cycle from selenite and iodide, respectively. As the latter amounts have an insignificant impact on plants, the untreated control was irrigated with water only at the same volume as the experimental treatments, with no K and Na addition. As can be observed in

Table 1. Beginning and end dates of crop cycle, as well as biofortification treatments.

Crop Cycle	Starting Date	First Se/I Treatment	Second Se/I Treatment	Third Se/I Treatment	Date of Leaf Cutting
First	2 November (sowing)	15 November	20 November	25 November	9 December
Second	9 December	25 December	30 December	4 January	24 January
Third	24 January	10 February	15 February	20 February	9 March

, the first cutting was conducted on 9 December which was the starting date of the second cycle, characterized by the same scheme of treatments as the first one. The second cutting was performed on 24 January which was the beginning of the third cycle. The biofortification treatments were conducted according to the same scheme in the three crop cycles.

Crop management was performed using sustainable management practices, including an organic preplant fertilization with N, P₂O₅, and K₂O (at a rate of 38, 10, and 30 kg ha⁻¹, respectively) and the control of fungal diseases and pests, when necessary, with copper oxychloride and azadirachtin treatments, respectively. During the three growing seasons, drip irrigation was activated when the soil available water at 10 cm depth dropped to 80%, and N, P₂O₅, and K₂O were supplied via fertigation during each crop cycle at a dose of 112, 30, and 90 kg ha⁻¹, respectively.

The harvests were conducted on 9 December, 24 January, and 9 March, when rocket leaves reached 6–7 cm length, cutting at 3–5 cm above the soil surface in order to leave the vegetative apex undamaged, thus allowing for more efficient regrowth.

As the research year did not have a significant effect on the examined variables and the latter were not significantly affected by the interaction between crop cycle and the biofortification treatment, the average values of the two trial years related to the main effects of the two experimental factors are presented.

2.2. Yield Determination

At each harvest, the following fresh yield parameters of perennial wall rocket were measured in all the experimental plots: total weight, the number of leaves, and mean leaf weight of 100 g samples.

2.3. Sample Preparation

Fresh leaf samples were randomly collected in each experimental plot and stored at −80 °C until the extractions and analyses of the antioxidant compounds (total phenols and the ascorbic acid), and antioxidant activity were performed. Dried plant material was ground to a powder in a grinder; then, a 500 mg sample was used for the extraction and determination of the mineral elements and nitrate.

2.4. Dry Matter

The dry matter content was determined gravimetrically after drying baby leaf samples to a constant weight at 70 °C in a forced air oven. The results were expressed as the weight/weight percentage of dry matter (% w/w).

2.5. Ascorbic Acid

The ascorbic acid content was assessed using the manual titration method based on the interaction of the ascorbic acid with sodium 2,6-dichlorophenol indophenolate (Tillman’s reagent) [48], with a detected limit of 4 mg L⁻¹.

2.6. Total Polyphenols (TPs)

The Folin–Ciocalteu colorimetric method was used for total polyphenol determination by utilizing 70% ethanol extracts of dry leaf homogenates [49]. The concentration of polyphenols was assessed using the absorption value of the Folin reagent–sample ethanol extract reaction mixture at 730 nm (Unico 2804 UV spectrophotometer, Suite E Dayton, NJ, USA). A solution of 0.02% gallic acid was used as an external standard, and all the results were expressed in mg of gallic acid equivalent per g of dry weight (mg GAE g⁻¹ d.w.). The detected limit was 1 µg mL⁻¹.

2.7. Total Antioxidant Activity (AOA)

To evaluate the total antioxidant activity of perennial wall rocket leaves, a 0.01 N KMnO₄ solution was titrated with the ethanol extracts of leaf homogenates until the solution became completely discoloured [49]. Gallic acid (PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany) was used as an external standard, and the values obtained were expressed in mg GAE g⁻¹ d.w.

2.8. Proteins

The crude protein content was measured via the Kjeldahl methodology, which is based on sample digestion with sulfuric acid, and the quantification of the ammonia content after reaction mixture alkalization was conducted [50]. The detected limit was 0.1 mg.

2.9. Soluble Solids

The determination of wall rocket leaf soluble solids was carried out using a portable digital refractometer, model DBR 35 (Sinergica Soluzioni s.r.l., Pescara, Italy), expressed in °Brix.

2.10. Nitrates

Nitrates were assessed using ion chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA) coupled to a conductivity detector, with an IonPac AG11-HC guard (4 × 50 mm) column and an IonPac AS11-HC analytical column (4 × 250 mm). The flow rate of KOH solution (1 to 50 mM gradient) was 1.5 mL min⁻¹. The detection limit reached 0.08 mg L⁻¹.

2.11. Colour Measurement

Leaf colour was measured with a portable colorimeter (CR-10 Chroma; Minolta, Osaka, Japan) with Cilluminant after calibration with a white standard calibration plate, referring to the CIELab components L, a, and b. The ‘L’ component represents the relative lightness ranging from 0 (black) to 100 (white); both ‘a’ and ‘b’ range from –60 to +60, i.e., ‘a’ from green to red and ‘b’ from blue to yellow.

2.12. Mineral Determination

D. tenuifolia’s leaf elemental profile, including Ca, K, Mg, P, Cu, Fe, Mn, and Zn, was determined in triplicate on dried, homogenized leaf samples using ICP-MS on the quadruple mass spectrometer Nexion 300D (Perkin Elmer Inc., Shelton, CT, USA), after the digestion of 1 g sample with sub-boiled HNO₃ (Fluka No. 02, 650 Sigma-Aldrich, Co., Saint Louis, MO, USA), 1:150 diluted with distilled deionized water in a Berghof SW-4 DAP-40 microwave digestion system (Berghof Products + Instruments Gmb H, 72, 800 Eningen, Germany). The analysis details were as follows: plasma power and argon flow, 1500 and 18 L min⁻¹, respectively; nebulizer argon flow, 0.98 L min⁻¹; and aux argon flow, 1.6 L min⁻¹. A sample introduction system was represented using the ESI ST PFA concentric nebulizer and the ESI PFA cyclonic spray chamber (Elemental Scientific Inc., Omaha, NE, USA) with the following parameters: injector, ESI Quartz 2.0 mm I.D.; sample flow, 637 L min⁻¹; standard internal flow, 84 L min⁻¹; and dwell time and acquisition mode, 10–100 ms with peak hopping for all the analytes. Rhodium 103 Rh was used as an internal standard to eliminate instability during the measurements. Quantitation was performed using an external standard (Merck IV, a multi-element standard solution); the Perkin–Elmer standard solution for P and all the standard curves were obtained at five different concentrations.

2.13. Selenium Content

The selenium content in rocket leaves was analyzed using the fluorimetric method previously described for tissues and biological fluids [51]. A total of 0.1 g of dried homogenized sample powder was digested via heating with a mixture of nitric and perchloric acids, according to the following sequence: at 120 °C for 1 h, 150 °C for 1 h, and 180 °C for 1 h. To reduce selenates (Se⁺⁶) to selenites (Se⁺⁴), a solution of 6 N HCl was used. Selenium concentration was obtained based on the fluorescence value of a complex between Se⁺⁴ and 2,3-diaminonaphtalene (hexane, 376 nm λ excitation; 519 nm λ emission). Each determination was performed in triplicate. The precision of the results was verified using the Mitsuba reference standard of Se-fortified stem powder for each determination, with a Se concentration of 1865 µg kg⁻¹ (Federal Scientific Vegetable Center). The detection limit was 0.8 ng.

2.14. Iodine Content

To determine I content, 0.15 g of lyophilized and homogenized leaves was extracted with 10 mL of distilled water and 2 mL of 25% tetramethylammonium hydroxide. The extracts were filtered and analyzed via ICP-MS [52]. The detection limit was 4 µg L⁻¹. An external multi-element standard solution (Merck IV) was used for each determination.

2.15. Statistical Analysis

The presented results are the mean values of three replicates per experimental treatment. The data were statistically processed using analysis of variance (ANOVA), and mean separation was performed through Duncan’s multiple range test at p < 0.05 using the SPSS software version 29 (IBM, Armonk, NY, USA).

3. Results and Discussion

3.1. Yield and Plant Performance

The wall rocket production is usually targeted to perform at least three cuttings of leaves [39,40]. As no significant interactions arose between the two experimental factors applied, the significant effects of crop cycle on wall rocket yield are presented as the means of the biofortification treatments and the untreated control. The first cycle provided the highest production, 2.12 and 2.4 times higher than those corresponding to the second and third ones, respectively, due to both higher leaf number per m² and mean weight (

Table 2. Effects of crop cycle and the biofortification treatment on yield parameters and plant dry matter of perennial wall rocket (means of the two-year data) (n = 3).

Treatment	Crop Surface (m2)	Yield (g m−2)	Number of Leaves per m2	Mean Leaf Weight (g)	Plant Dry Matter (g m−2)
Crop cycle (means of 4 values: I, Se, Se + I, control)
Autumn	12	1536.6 ± 319.8 a	1442.8 ± 300.8 a	1.08 ± 0.13 a	158.7 ± 42.6 a
Autumn–winter	12	724.2 ± 163.7 b	1213.8 ± 208.8 b	0.59 ± 0.05 b	76.4 ± 23.4 b
Winter	12	640.0 ± 98.6 b	1266.9 ± 179.2 b	0.51 ± 0.02 b	66.1 ± 14.5 c
Biofortification treatment (means of the 3 crop cycles)
Control	9	743.9 ± 347.2 c	987.9 ± 63.3 c	0.75 ± 0.34 ab	67.0 ± 32.1 c
I	9	893.5 ± 357.6 b	1320.1 ± 146.8 b	0.66 ± 0.21 b	89.4 ± 36.2 b
Se	9	1020.7 ± 446.5 a	1391.0 ± 221.0 b	0.71 ± 0.22 ab	111.5 ± 47.5 a
Se + I	9	1209.6 ± 589.0 a	1532.3 ± 147.2 a	0.77 ± 0.31 a	133.7± 62.4 a

Within each column, values followed by different letters are statistically different according to Duncan’s test at p ≤ 0.05.

). Plant dry matter per m² showed a similar trend to yield.

The significant effects of the biofortification treatment on the yield and growth parameters are reported only as mean values of the three crop cycles, as no significant interactions arose between the two experimental factors applied (Table 2). In this respect, the combined application of Se and I resulted in the highest yield as a consequence of the highest leaf number and mean weight, exceeding the lowest production associated with the untreated control by 62.6%, the single I supply by 20.1%, and the Se treatment by 37.2%. The synergetic effect of Se and I on plant yield has been previously described in chervil plants, with a more pronounced growth stimulation effect in roots than shouts [15]. A similar trend to the yield was recorded for plant dry matter per m². The results are in accordance with the growth stimulation effect of Se and I described in different agricultural crops. At present, the beneficial effect of low iodine doses on plant yield is explained by the ability of I to regulate the expression of various genes and to affect the activity and structure of various proteins as a result of iodination [53]. Low doses of Se are known to maintain cell structure and improve water availability and nitrogen assimilation [54], which is consistent with the detected beneficial effect of this element on D. tenuifolia in the present research.

Joint Se/I biofortification had a growth stimulation effect on perennial wall rocket, differing from other species which were not significantly affected in terms of plant production. Indeed, carrot [10], potato [12], pumpkin [16], and Indian mustard [13] did not show a yield increase under joint Se/I biofortification. Overall, the results of the present research revealed significant growth stimulation effects of both single and combined applications of Se and I on wall rocket production, suggesting high prospects of this new technology.

3.2. Quality, Colour, and Antioxidant Parameters

Overall, the biochemical parameters and mineral element contents of wall rocket leaves, presented using the same pattern as for yield and plant growth variables, were significantly affected by both crop cycle and Se/I biofortification. In this respect, only the dry residue, soluble solids, colour components L, a, and b (

Table 3. Effects of crop cycle and the biofortification treatment on rocket leaf quality and colour (means of the two-year data) (n = 3).

Treatment	Crop Surface	Dry Matter	Soluble Solids	Colour Parameters
	(m2)	(%)	(°Brix)	L	a	b
Crop cycle (means of 4 values: I, Se, Se + I, control)
Autumn	12	10.2 ± 0.8	5.7 ± 0.5	38.5 ± 1.9	−13.4 ± 1.1	20.0 ± 1.8
Autumn–winter	12	10.4 ± 1.1	5.7 ± 0.6	39.5 ± 1.0	−12.2 ± 1.0	22.6 ± 1.3
Winter	12	10.2 ± 0.9	5.8 ± 0.6	40.7 ± 1.0	−13.2 ± 1.0	21.8 ± 1.1
		n.s.	n.s.	n.s.	n.s.	n.s.
Biofortification treatment (means of the 3 crop cycles)
Control	9	8.97 ± 0.16 b	4.90 ± 0.23 b	37.9 ± 2.6	−12.4 ± 1.0	23.0 ± 1.6
I	9	9.99 ± 0.29 ab	5.95 ± 0.23 a	40.4 ± 1.0	−13.2 ± 1.4	20.9 ± 1.9
Se	9	10.97 ± 0.20 a	6.00 ± 0.20 a	40.1 ± 1.6	−13.4 ± 0.9	21.3 ± 1.4
I + Se	9	11.14 ± 0.42 a	6.08 ± 0.17 a	39.8 ± 1.2	−12.7 ± 0.9	20.7 ± 1.6
				n.s.	n.s.	n.s.

Within each column, values followed by different letters are statistically different according to Duncan’s test at p ≤ 0.05.

), and vitamin C content (

Table 4. Effects of crop cycle and the biofortification treatment on rocket leaf antioxidant compounds and activity (means of the two-year data) (n = 3).

Treatment	Crop Surface	Total Antioxidant Activity	Total Polyphenols	Vitamin C
	(m2)	(mg-GAE g−1 d.w.)	(mg-GAE g−1 d.w.)	(g 100 g−1 f.w.)
Crop cycle (means of 4 values: I, Se, Se + I, control)
Autumn	12	25.5 ± 1.9 b	13.5 ± 1.7 b	20.8 ± 2.2 a
Autumn-winter	12	25.1 ± 2.2 b	14.4 ± 1.3 ab	21.1 ± 2.2 a
Winter	12	27.2 ± 3.4 a	15.3 ± 1.8 a	21.4 ± 2.0 a
Biofortification treatment (means of the 3 crop cycles)
Control	9	22.9 ± 1.1 b	12.9 ± 1.0 b	18.5 ± 0.9 a
I	9	25.0 ± 1.6 b	13.5 ± 1.2 b	20.3 ± 0.9 a
Se	9	27.5 ± 1.9 a	15.4 ± 1.3 a	22.4 ± 1.0 a
Se + I	9	28.4 ± 1.8 a	15.8 ± 1.5 a	23.2 ± 1.0 a

Within each column, values followed by different letters are statistically different according to Duncan’s test at p ≤ 0.05.

) were not statistically affected by crop cycle (Table 3); moreover, the colour parameters did not significantly differ between the Se/I biofortification treatments (Table 3).

The Se/I supply resulted in the significant increase in dry residue, with the highest values recorded under the Se + I and Se applications which exceeded the untreated control by 24% and 22%, respectively (Table 3). Furthermore, the soluble solids value was the highest in the Se + I treatment, showing a 1.24-fold increase compared to the control (Table 3).

The protein content decreased from the first to the second and third crop cycle by 19.7% and 23.4%, respectively, displaying a similar trend as yield (Figure 1).

On the contrary, both Se and I treatments provided similar significant increases in the protein content, by 36–48%, 20–67%, and 54–61% under selenium, iodine, and Se/I joint applications, respectively. The recorded beneficial effect of Se and I on protein accumulation relates to the well-known participation of selenium in nitrogen metabolism and a stimulation of protein biosynthesis by Se [55] and I [23].

The response of plants to environmental stresses is closely connected with the intensity of biosynthesis of secondary metabolites participating in the antioxidant defence [56]. In this respect, the total antioxidant activity and polyphenol content recorded in the third crop cycle were higher than those detected in the two earlier cycles by 2 mg GAE g⁻¹ d.w. and 13.3%, respectively, which confirms the enhancement of plant oxidative stress in the latest cycle (Table 4).

According to the obtained data (Table 4), the Se/I supply, especially under joint application, resulted in a significant increase in both the polyphenol content and the total antioxidant activity, suggesting the importance role of the mentioned microelements in the antioxidant defence. In general, wall rocket is known to be a good natural source of polyphenols with the prevalence of quercetin derivatives [38,57,58]. The remarkable antioxidant properties of quercetin and its protection against cardiovascular diseases and cancer [59] suggest the importance of the significant increase in the polyphenol content in this study; under the Se and Se + I supply, it increased by approximately 1.2-fold, compared to both the I treatment and control (Table 4). The total antioxidant activity of wall rocket leaves was higher upon Se + I and Se applications, exceeding the untreated control by 23.6% and 20.2%, respectively (Table 4). The impact of joint biofortification with I and Se recorded in other crops are consistent with the results of the present research. Indeed, the foliar Se/I biofortification of Indian mustard resulted in more than a two-fold increase in flavonoid content but slightly affected the ascorbic acid level [13]. On the contrary, only a 12.5% increase was recorded in the total antioxidant activity and polyphenol content in chickpea due to joint Se/I biofortification [14] and a 12% rise in polyphenols in lettuce [9], confirming the importance of genetic factors for the maintenance of plant antioxidant status.

3.3. Mineral Composition

D. tenuifolia is known to be a good source of Ca, K, Mg, and Fe [60]. In this respect, the sequential cutting of leaves may be considered as an external oxidant stress affecting both the yield and antioxidant status of wall rocket (Table 2 and Table 4), as well as causing a mineral imbalance in plant tissues (

Table 5. Effects of crop cycle and the biofortification treatment on the macroelement content in rocket leaves (means of the two-year data) (n = 3).

Treatment	Ca (g kg−1 d.w.)	Mg (g kg−1 d.w.)	NO3− (mg kg−1 f.w.)	P (g kg−1 d.w.)
Crop cycle (means of 4 values: I, Se, Se + I, control)
Autumn	27.4 ± 2.7 b	3.45 ± 0.32 b	5150 ± 408 a	2.84 ± 0.40 b
Autumn-winter	29.0 ± 3.7 ab	3.59 ± 0.37 ab	5079 ± 342 a	2.95 ± 0.42 ab
Winter	31.1 ± 4.2 a	3.81 ± 0.48 a	4206 ± 287 b	3.11 ± 0.45 a
Biofortification treatment (means of the 3 crop cycles)
Control	25.7 ± 1.6 b	3.24 ± 0.17 b	5260 ± 586 a	2.52 ± 0.16 b
I	26.6 ± 1.8 b	3.33 ± 0.12 b	4763 ± 443 b	2.65 ± 0.15 b
Se	31.6 ± 2.6 a	3.88 ± 0.26 a	4625 ± 478 b	3.29 ± 0.19 a
Se + I	32.7 ± 1.8 a	4.01 ± 0.31 a	4597 ± 494 b	3.39 ± 0.20 a

Within each column, values followed by different letters are statistically different according to Duncan’s test at p ≤ 0.05.

and

Table 6. Effects of crop cycle and the biofortification treatment on the microelement content in rocket leaves (means of the two-year data) (n = 3).

Treatment	Zn (mg kg−1 d.w.)	Fe (mg kg−1 d.w.)	Cu (mg kg−1 d.w.)	Mn (mg kg−1 d.w.)
Crop cycle (means of 4 values: I, Se, Se + I, control)
Autumn	11.9 ± 1.8 a	239.2 ± 132.7 a	15.9 ± 7.5 a	10.8 ± 4.5 b
Autumn-winter	11.9 ± 1.9 a	233.0 ± 107.6 a	14.9 ± 7.9 a	16.6 ± 2.6 a
Winter	14.1 ± 2.4 a	96.5 ± 21.4 b	6.6 ± 1.8 b	7.0 ± 1.9 c
Biofortification treatment (means of the 3 crop cycles)
Control	9.7 ± 0.8 b	77.0 ± 20.8 d	5.9 ± 1.3 c	15.6 ± 4.7 a
I	12.9 ± 1.3 a	173.3 ± 65.9 c	19.0 ± 7.8 b	11.8 ± 4.9 b
Se	13.7 ± 2.0 a	225.5 ± 113.7 b	8.4 ± 2.1 a	8.8 ± 4.3 c
Se + I	14.0 ± 1.5 a	282.4 ± 129.9 a	16.4 ± 6.7 c	9.6 ± 4.1 c

Within each column, values followed by different letters are statistically different according to Duncan’s test at p ≤ 0.05.

).

Among the examined macroelements, Ca, Mg, and P showed significant increased levels in the third crop cycle, exceeding those of the first crop cycle by 13.6%, 10.4%, and 9.5%, respectively, regardless of the Se/I application (Table 5).

In contrast, the decreases in potassium (Figure 2) and nitrate content (Table 5) from the first to the third crop cycle were 40% and 21.5%, respectively.

The changes in potassium content in the wall rocket leaves from the first to the third cycle are consistent with the lowest yield and the highest antioxidant status of plants recorded in the third cycle. The mentioned dynamics suggest the important role of K and nitrogen in plant growth, as well as the oxidant stress enhancement in the leaves harvested in the latest cycle. In this respect, Se/I supply to wall rocket plants was extremely beneficial for improving the potassium accumulation levels.

Considering the negligible amount of K supplied by the potassium iodide treatment, the significant effect of Se has arisen on the increase in potassium level which has not been significantly affected by I application.

A general picture of the Se/I effect on macroelement accumulation in wall rocket, shown in Figure 3, confirms the beneficial effect of Se/I treatments for improving the nutritional value of plants, indicating a much lower effect of iodine compared to Se and Se + I supply.

In this respect, the mean values of calcium content in all three crop cycles were 1.2-fold higher under the Se + I and Se treatments, compared to the I treatment and control (Figure 3). The same pattern was observed for Mg, whose contents under the Se + I and Se treatments exceeded those relevant to I supply and control by 23.8% and 19.8%, respectively. The phosphorus concentration under the Se + I and Se treatments was about 1.3 times higher compared to the I treatment and the untreated control, while the most significant effect was recorded for K.

To date, the effect of Se and I supply on mineral composition of plants has been studied rather sporadically, thus impeding the creation of a univocal frame of changes in the element levels [60,61]. In this respect, the perennial wall rocket mineral profile recorded under the biofortification treatments in the present research is particularly interesting (Table 5).

A different trend was observed for nitrate content which showed the highest value in the wall rocket control leaves, exceeding the average of the biofortification treatments by about 12%. In this respect, it is worth mentioning that iodine treatment to radish did not change the nitrate content [62] but increased the free amino acid concentration. According to the literature reports, Se supply decreases nitrate levels by enhancing the activity of nitrate reductase [13].

Wall rocket is known to be a hyper-accumulator of nitrates capable of accumulating up to 9000 mg kg⁻¹ f.w. or even more depending on growth conditions [63,64]. The maximum threshold of nitrate content in wall rocket leaves according to the European Commission Regulation [65] is 6000 mg kg⁻¹ f.w. in spring–summer and 7000 mg kg⁻¹ in autumn–winter, while the acceptable daily intake (ADI) of nitrates should not exceed 3.7 mg kg⁻¹ bw⁻¹ day⁻¹. In general, the high consumption of nitrates may cause health risks due to the possibility of carcinogenic nitrosamine formation in the stomach under the presence of secondary amines and methemoglobin synthesis which hamper oxygen accumulation [66]. However, nitrate is also highly valuable for human health, as it prevents hypertension and protects human organism against cardiovascular diseases [67,68] via the regulation of gene expressions of proteins and enzymes involved in nitric oxide synthesis. The obtained results indicate a significant decrease in the nitrate levels both under Se and I supply, which is in accordance with the Se influence on nitrogen metabolism, usually decreasing the content of this ion in plants [13,69], and a competition between iodine and nitrates in plant nutrient uptake [70]. However, the high levels of natural antioxidants present in vegetables and especially in wall rocket guarantee the restriction of nitrosamine formation, thus diminishing health risks and providing conditions for beneficial nitrogen oxide formation [71].

Regarding the microelements in rocket leaves (Figure 4), Se concentration reached its peak in the third cycle, exceeding the values recorded in both the first and second cycles by approximately 16%. The same pattern was observed for iodine content, with the third cycle showing a 60% higher concentration than the first and second cycles.

The data presented in Figure 4A indicate a slight stimulation effect of I on Se accumulation both in the control and Se-treated plants. The similar beneficial effect of Se on iodine content was recorded in all three crop cycles. Controversial results of Se/I interaction in plants, reflecting the possibility of both synergistic and antagonistic relationships between the two microelements [1,11,49], suggest the need for further investigations aimed at revealing the factors affecting the related phenomenon.

In the present study, the selenium concentration in rocket leaves was the highest under the Se + I treatment, followed by the Se and I applications, with the lowest value corresponding to the untreated control. The biofortification levels of Se was equal to 50 in the case of joint Se/I supply, exceeded 44 upon single Se spraying, and reached 4.5 under the I treatment without endogenous Se. Iodine showed the peaked content in rocket leaves supplied with Se + I, followed by the I treatment, whereas it was not detected at all upon Se application and in the untreated control.

In general, the increase in Se/I accumulation in the leaves grown in the third crop cycle, compared to the second and the first one, may be related to the improvement of the plant’s antioxidant status under Se/I supply.

Changes in Zn, Fe, Cu, and Mn contents as a result of Se/I biofortification in different crop cycles are presented in Table 6 and Figure 5. In this respect, the zinc accumulation in the leaves harvested in the third cycle was about 1.2 times higher compared to those detected in the first and second cycles. Contrastingly, iron concentration significantly decreased in the third cycle, showing 2.5 times lower values than those measured in the first and second cycles. The copper content displayed a similar trend to iron, with the lowest value recorded in the leaves during the third cycle, 57% lower than those related to the first and second cycles. The manganese content was the highest in the leaves during the second cycle, followed by the first and the third cycles.

Surprisingly, the Se/I biofortification of perennial wall rocket resulted in the increased contents of both the macroelements and Zn, Fe, and Cu (Table 6, Figure 6).

Indeed, Zn and Fe concentrations were higher under the Se + I treatment, exceeding the untreated control by 44% and 370%, respectively. The copper content reached its maximum in the I treatment, 3.2 times higher than the control. A different pattern was observed for Mn, as the untreated leaves showed the highest concentration, exceeding the biofortification treatments by 30% to 43%.

The beneficial effect of single Se and I supply on mineral composition of wall rocket leaves was in agreement with previous findings in other plant species [72,73], but the joint Se/I effect on D. tenuifolia element accumulation was much higher than those recorded in different crops [8,13,15].

3.4. Correlations Between the Variables

The relationship between wall rocket weight, mineral composition, and antioxidant status confirmed the importance of Se/I supply on crop yield and on the nutritional value of the product (

Table 7. Coefficients of correlation between the examined parameters of wall rocket leaves (n = 12).

	Ca	Mg	K	NO3	Se	I	Zn	Fe	Cu	Mn	DM	SS	Protein	AOA	TP	AA	Yield
P	0.893 a	0.903 a	0.367	−0.553 f	0.925 a	0.232	0.703 c	0.420	−0.046	−0.509	0.826 a	0.727 c	0.603 f	0.797 a	0.766 a	0.827 a	0.117
	Ca	0.848 a	0.161	−0.582 f	0.838 a	0.273	0.751 b	0.255	−0.138	−0.512	0.706 c	0.626 e	0.453	0.800 a	0.762 a	0.782 a	−0.041
		Mg	0.221	−0.583 f	0.842 a	0.258	0.717 c	0.250	−0.124	−0.492	0.739 b	0.636 e	0.491	0.797 a	0.814 a	0.747 b	−0.009
			K	0.361	0.617 b	−0.053	−0.006	0.762 a	0.431	0.177	0.528	0.345	0.688 d	0.182	0.140	0.385	0.617 e
				NO3	−0.446	−0.391	−0.733 b	0.161	0.220	0.733 b	−0.442	−0.510	−0.175	−0.600 f	−0.603 f	−0.500	0.267
					Se	0.168	0.656 e	0.509	0.024	−0.503	0.872 a	0.686 d	0.753 b	0.815 a	0.750 b	0.842 a	0.291
						I	0.515	0.159	0.443	−0.300	0.324	0.515	0.270	0.415	0.247	0.345	0.08
							Zn	0.178	0.035	−0.734 b	0.664 d	0.747 b	0.504	0.830 a	0.710 c	0.670 d	0.005
								Fe	0.615 f	−0.006	0.612 f	0.501	0.720 c	0.223	0.214	0.504	0.602 f
									Cu	0.161	0.284	0.343	0.470	−0.032	−0.122	0.195	0.473
										Mn	−0.431	−0.506	−0.462	−0.622 f	−0.484	−0.487	−0.214
											DM	0.837 a	0.783 a	0.760 a	0.620 f	0.836 a	0.271
												SS	0.721 c	0.727 c	0.581 f	0.747 b	0.284
													Protein	0.580 f	0.402	0.705 c	0.739 b
														AOA	0.751 b	0.788 a	0.100
															TP	0.619 f	−0.123
																AA	0.244

(a) p < 0.001; (b) 0.002; (c) p < 0.005; (d) p < 0.01; (e) p < 0.02; (f) p < 0.05; DM: dry matter; SS: soluble solids; AOA: total antioxidant activity; TP: polyphenols; AA: ascorbic acid.

).

The role of selenium in activating the antioxidant metabolism in plants was previously demonstrated in different plants [74,75] and is reflected by a strong correlation between Se, polyphenol, and ascorbic acid content, as well as the total antioxidant activity in wall rocket leaves (r > 0.815; p < 0.001). According to the obtained data, the biofortification of wall rocket with Se and I is characterized by significant correlations of Se both with the antioxidant parameters and with the contents of Ca, P, and Mg (Table 7, Figure 3).

A more complex interaction was revealed between Se and plant minerals. Indeed, the foliar supply of tea plants with glucosamin selenium was shown to increase macro- and microelement accumulation along with the enhancement of plant antioxidant status [76]. On the contrary, the Se treatment of maize resulted in Ca and P level increase, K concentration decrease, and no effects on Mg [77]. Se biofortification did not significantly affect the mineral content in oats [78], whereas Zinc, Ca, and Mg significantly increased under Se foliar supply in wheat plants [79]. In our research, the conditions of wall rocket Se/I biofortification greatly fostered the plant quality in terms of both antioxidant and mineral composition, which are reportedly affected by selenium dose, the method of supply, and genetic and environmental factors [22,79].

The data presented in Table 7 show that no significant correlations between iodine, Se, and other elements arose, suggesting the different mechanisms of Se and I uptake by plants. Indeed, KI is absorbed via chloride and Na-K/Cl transporters, while selenate via sulphate transporters [1]. In general, the beneficial effect of Se/I biofortification on wall rocket yield is connected with the positive correlation between plant yield, potassium, and proteins (Table 7); i.e., Se increased protein and K content, thus promoting perennial wall rocket growth.

3.5. Mineral Consumption Levels

The significant increase in antioxidant properties as well as macro- and microelement contents under Se/I biofortification represents an interesting outcome with the perspective to produce functional food with innovative characteristics (

Table 8. Consumption of mineral elements corresponding to 50 g of fresh wall rocket leaves biofortified with Se + I, compared to the dietary allowance level (RDA), in %.

Element	RDA (mg)	Treatment
		Control	Iodine	Selenium	Iodine + Selenium
Fe	8	5.7 (3.9) *	14.2 (6.3)	22.3 (6.6)	24.0 (7.3)
Cu	2	1.8 (1.1)	6.1 (2.2)	2.7 (1.5)	5.3 (1.9)
Zn	10	0.5 (0.6)	0.6 (0.7)	0.7 (0.8)	0.7 (0.8)
Mn	2.3	3.9 (2.1)	2.1 (1.7)	1.6 (1.1)	1.7 (1.3)
Se	0.070	1.8 (2.1)	7.9 (9.4)	78.9 (93.4)	88.8 (100)
I	0.150	0	23.4 (37.7)	0	25.9 (41.6)
Ca	1000	14.4 (13.4)	12.7 (14.1)	14.7 (17.1)	15.0 (17.7)
K	3510	6.2 (3.3)	6.7 (4.0)	8.1 (5.1)	9.0 (5.5)
Mg	350	4.5 (4.7)	4.6 (5.3)	5.3 (5.9)	5.4 (6.2)
P	800	1.5 (1.6)	1.6 (1.7)	2.0 (2.2)	2.0 (2.2)

* Values in parenthesis refer to the third crop cycle.

). The consumption levels of macro- and microelements associated with 50 g of fresh perennial wall rocket leaves suggest the importance of Se/I biofortified plants as a remarkable source of Se, I, Ca, and K. Iron ingestion, attaining 24% of RDA, should not be taken into consideration as only 7–9% of non-heme iron is absorbed by leafy vegetables [80]. Despite the great differences in Fe, Cu, Mn, Se, and I between the first and the third crop cycles, Se + I biofortified plants grown in the first crop cycle can provide, referring to RDA, up to 24% of Fe, 9.0% of K, 15% of Ca, 25.9% of I, and 88.8% of Se (Table 8). Furthermore, Cu, Zn, Mn, Mg, and P contents in wall rocket leaves may not be taken into account due to their low concentrations, providing less than 5% of the corresponding RDA values. On the contrary, significantly higher consumption levels of Se and I can be obtained from the third crop cycle, with values reaching 100% and 41.6% of RDA, respectively.

In general, it may be inferred that the obtained Se/I biofortified wall rocket plants may be useful for decreasing the risks of cardiovascular diseases, osteoporosis, high blood pressure due to the high levels of Ca, K, and Se [73,81], as well as for enhancing both human organism immunity and Se/I status.

4. Conclusions

The foliar Se/I biofortification of Diplotaxis tenuifolia represents an interesting technology to boost yield, antioxidant status, Se, and I, as well as Ca, Mg, K, P, Fe, Cu, and Zn accumulation in the leaves of this species. The mentioned treatments allowed us to create a new functional food with high Se and I levels, useful to prevent and cure human Se/I deficiency, and protect against cardiovascular diseases and cancer due to the high levels of Se, I, K, and Ca. The present research has brought the perspective of valorising perennial wall rocket in terms of the industrial production of health beneficial products under sustainable management.