Plant Physiology and Biochemistry 206 (2024) 108283 Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy Selenium biofortification of microgreens: Influence on phytochemicals, pigments and nutrients Marcia Viltres-Portales a, b, 1, María-Jesús Sánchez-Martín a, *, 1, Mercè Llugany c, Roberto Boada a, Manuel Valiente a a GTS Research Group, Department of Chemistry, Faculty of Science, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain b Institute of Materials Science and Technology, Universidad de La Habana, Zapata y G, Vedado, Plaza, 10400, La Habana, Cuba c Plant Physiology Group (BABVE), Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: Kale (Brassica oleracea L. var. sabellica L.), kohlrabi (Brassica oleracea L. var. gongylodes L.) and wheat (Triticum Kale aestivum L. cv. Bancal) microgreens were cultivated in presence of selenium 20 μmol L− 1 as sodium selenite and Kohlrabi sodium selenate mixture. The influence of this biofortification process was evaluated in terms of biomass pro­ Wheat duction, total Se, macro- and micronutrients concentration, polyphenols, antioxidant activity, chlorophylls and Microgreens carotenoids levels and total soluble proteins content. The results obtained have shown a significant concentration Selenium Biofortification of total Se in the biofortified microgreens of kale (133 μg Se⋅g− 1 DW) and kohlrabi (127 μg Se⋅g− 1 DW) higher than that obtained for wheat (28 μg Se⋅g− 1 DW). The Se uptake in all the species did not produce oxidative damage to the plants reflected in the bioactive compounds, antioxidant capacity or pigments concentration. These Se-enriched microgreens may contribute to the recommended intake of this nutrient in human diet as to overcome Se-deficiency. 1. Introduction In addition to the short growing time, microgreens also require less space, water, and substrate for large-scale production than mature Microgreens are young seedlings that are typically harvested 1–3 plants. The production of microgreens ranges from innovative hydro­ weeks after germination when they are only a few centimeters tall and ponic techniques to improved greenhouses and vertical farms where the first true leaf has appeared (Partap et al., 2023). These small plants virtually no pesticides or herbicides are needed (Kyriacou et al., 2016). are produced from the seeds of vegetables, herbs, grains, ornamental, or These crops can therefore be considered environmentally friendly and even wild species. In recent years, microgreens have gained popularity can be produced in densely populated areas. With these advantages and as a new culinary trend due to their variety of flavors, vibrant colors, nutraceutical values, microgreens are promising targets for increasing appearance, and textures, allowing them to be use in salads, soups, or the level of essential mineral nutrients in plant foods through bio­ many other dishes. Furthermore, due to their high nutritional value, fortification techniques. microgreens have earned the title of “superfood” or “functional foods” as Biofortification is a process aimed to improve the nutritional value of they contain considerably higher concentrations of phytonutrients and crops by increasing the concentration of essential micronutrients in secondary metabolites, such as amino acids, enzymes, pigments, vita­ edible portions without sacrificing agronomic characteristics such as mins, polyphenols, and antioxidants, than their mature plant counter­ yield, or resistance to pests and drought (Dhaliwal et al., 2022). Among parts (Nair and Lekshmi, 2023). This unusual combination of high levels the different strategies for obtaining biofortified crops are agronomic or of macro- and micronutrients is related to their important role in health, genetic approaches. The latter through conventional breeding, where functioning as antioxidants, and immunomodulators, against inflam­ plant varieties with higher nutrient content are selected and crossed, or mation, obesity, cardiovascular diseases, cancer, diabetes, among others using transgenic programs that involve biotechnology studies such as (Sharma et al., 2022). the genetic modification of species to obtain a plant with specific * Corresponding author. E-mail address: mariajesus.sanchez@uab.cat (M.-J. Sánchez-Martín). 1 Shared co-first authorship. https://doi.org/10.1016/j.plaphy.2023.108283 Received 2 October 2023; Received in revised form 11 December 2023; Accepted 13 December 2023 Available online 19 December 2023 0981-9428/© 2023 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 characteristics (Sheoran et al., 2022). However, agronomic bio­ acetone were acquired in Scharlab (Barcelona, Spain). Gallic acid and fortification has several advantages and has been more widely applied 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) were purchased from due to its cost-effectiveness and ease of scaling (Teklu et al., 2023). Sigma-Merck (Schnelldorf, Germany). Other approaches, but addressed to a lesser extent, are biofortification through nanotechnology, where nanomaterials are applied to plants 2.2. Plant material and growth conditions alone or as a component of conventional fertilizers (e.g. Zn, Fe or gra­ phene nanoparticles), and green technologies, which involve the use of The experiment was carried out according to a randomized design in microorganisms to improve the nutrient status of the soil and the a factorial arrangement (2 × 3), with two concentrations (0 or 20 μmol accessibility of nutrients to plants (Dhaliwal et al., 2022). The success of L− 1) of a mixture of Na2SeO3 and Na2SeO4 (1/1, v/v) in tap water and biofortification programs depends on a combination of factors. These three species of microgreens (Ramos et al., 2011; Woch and include the availability of highly nutritious crop varieties adapted to Hawrylak-Nowak, 2019). Seeds of kale (Brassica oleracea L. var. sabellica local conditions, strategies to promote awareness and acceptance among L.- InstaGreen SL, Spain), kohlrabi (Brassica oleracea L. var. gongylodes L. communities, address regulatory issues, ensure sustainable production, - InstaGreen SL, Spain) and wheat (Triticum aestivum L. cv. Bancal- Fitó S. and foster public-private partnerships to facilitate widespread adoption A., Spain), were sown in cellulose sheets (7.5 × 11.5 cm) on plastic cups of biofortified crops (Van Ginkel and Cherfas, 2023). (10 × 14.5 × 5 cm) distributed in trays with a capacity for 40 cups. The Selenium (Se) is an essential trace element for humans and a plants were exposed to the solution with or without Se for 15 min twice a component of biologically important Se-proteins, such as antioxidant day. The study was carried out in a controlled environment of temper­ enzymes (Guardado-Félix et al., 2020). However, Se-deficiency in diets ature (25.8 ± 0.6 ◦ C) and relative humidity (59 ± 7 %) and considering currently affects 15 % of the world’s population, and its prevalence may a photoperiod of 16 h day/8 h night with a light intensity of 35 μmol increase with climate change (Schiavon et al., 2020). One of the solu­ m− 2⋅s− 1. The average of pH and conductivity value of the solutions used tions aimed at increasing the Se content in foods produced in was 7.6 ± 0.1 and 1.5 ± 0.3 mS cm− 1, respectively. After the specific Se-deficient areas is the biofortification of plants with this element. growth period for each microgreen (Table 1), the shoots were cut at a Biofortification of crops with this nutrient in open field is generally height of ⁓1.0 cm from the cellulose sheet. The growth of microgreens achieved through soil fertilization or foliar application of Se treatments. was analyzed to confirm the effects of different concentrations of Se. The most widely used Se compounds are inorganic Se salts (e.g., sodium Biomass yield (g per cup) was calculated in terms of fresh weight (FW) selenite and/or sodium selenate) due to their low cost and because and dry weight (DW). To determine DW, the plants were dried in an plants can transform these inorganic forms of Se into bioavailable oven at 50 ◦ C until constant weight. The experiment was repeated twice. Se-amino acids (Trippe and Pilon-Smits, 2021). These chemical forms of Se are involved in the maintenance of the immune system, regulation of 2.3. Total selenium, macro- and micronutrients thyroid function, cognitive function of the brain, antioxidant, and detoxification capacity, anticancer, and antiviral effects (Zhou et al., The dry plant material (200 mg) was digested with 10 mL of a 2020). Therefore, the combination of Se with bioactive compounds and mixture of HNO3/H2O2 (7:3, v/v) in a closed vessel of HP500 PFA at phytochemicals in microgreens is considered a new trend in the devel­ 180 ◦ C and 1.9 atm for 45 min using a microwave digestion system opment of functional foods (Islam et al., 2020; Mezeyová et al., 2022; (Mars 5, CEM, USA). The digested samples were filtered using 0.22 μm Newman et al., 2021; Pannico et al., 2020). Phenolic compounds, ca­ syringe filters and diluted until 3 % HNO3. The samples were analyzed rotenoids, and other secondary metabolites with antioxidant capacity for Se, sulfur (S), sodium (Na), potassium (K), phosphorus (P), calcium can scavenge free radicals and protect against high oxidative stress and (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), zinc related diseases. The high bioavailability of these compounds in (Zn), and boron (B) by inductively coupled plasma mass spectrometry microgreens may have anti-cancer, anti-microbial, anti-inflammatory, (ICP-MS) using an Agilent 7900 ICP-MS system (Agilent Technologies, and antidiabetic properties (Zhang et al., 2021). Inc., USA). The aim of the present study is the production of microgreens bio­ fortified with Se suitable for human consumption. To this end, three microgreens (kale, kohlrabi, and wheat) were selected based on their 2.4. Total polyphenolic compounds nutritional values and high demand in the food market. These plants were exposed to the combination of selenite and selenate ions The extraction of phenolic compounds was carried out following the throughout their growth time, since it is well known that selenate is method reported by Newman et al. with some modifications (Newman more easily transported from roots to shoots where it can accumulate et al., 2021). For extraction, 0.5 g of fresh weight microgreen samples but selenite metabolism to bioavailable organic species is faster and were mixed with 5 mL of methanol/water (80/20, v/v), stirred for 2 h in consumes less energy for plants. In previous works, the mixture of both the dark, sonicated in an ice bath for 15 min and then centrifuged at species resulted in a modulation of these two important parameters: the 2200 rpm for 10 min. The supernatant was collected, and the extraction accumulation and the toxic effect of each specie separately and the in­ process was repeated. Supernatants were combined and stored at − 20 ◦ C crease in the production of desired selenoamino acids in the consumable until analysis. The analysis of total polyphenolic compounds (TPC) was parts of the plants. Other analyses focused on the Se uptake and its in­ carried out according to the procedure of Singleton et al. (1999) with the fluence on macro- and micronutrients levels and on the bioactive com­ Folin–Ciocalteau reagent. In this case, 100 μL of the plant extracts and pounds, pigments and total soluble proteins produced by microgreens. Table 1 2. Materials and methods Cultivation characteristics of the microgreen species. Common Scientific name Density of seeds Cultivation days 2.1. Chemicals name per cup (g) in: Darkness Light Sodium selenate (Na2SeO4, 98 %), nitric acid (HNO3, 69 %) and 6- Kale Brassica oleracea L. var. 2.0 5 3 hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox re­ sabellica L. agent) were obtained from Thermo Fisher Scientific Inc. (Barcelona, Kohlrabi Brassica oleracea. L. var. 2.3 7 3 Spain). Sodium selenite (Na2SeO3), Folin–Ciocalteau reagent and so­ gongylodes L. dium carbonate (Na2CO3) were from VWR International (Barcelona, Wheat Triticum aestivum L. cv. 20 5 8 Bancal Spain). Hydrogen peroxide (H2O2, 30 %), methanol (HPLC grade) and 2 M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 500 μL of the Folin–Ciocalteau reagent (0.2 N) were mixed and incu­ 2.8. Data analysis bated for 5 min in the dark at room temperature. Then, 400 μL of sodium carbonate (75 g L− 1) was added and the solution was incubated again for All analyses were performed for the two experiments with three 2 h under the same conditions as before. Finally, 200 μL of the solution replicates of each microgreen treated with and without Se (n = 6). Data was added to a 96-well microplate and the absorbance was measured at analysis was performed with OriginPro 9.0 software using one-way 760 nm with an Infinite M200Pro microplate reader (Tecan Trading AG, analysis of variance (ANOVA) to determine the differences between Switzerland). TPC were determined using a gallic acid standard curve treatments. The comparison of means was done by the Tukey test at the (0–150 mg L− 1) and were expressed in mg of gallic acid equivalents 5% significance level (p-value ≤0.05). (GAE) per gram of FW. 3. Results and discussion 2.5. Antioxidant activity 3.1. Biomass yield Antioxidant activity was measured using the radical 2,2-diphenyl-1- The observed differences in biomass yield between the different picrylhydrazyl (DPPH) as described by Brand-Williams, Cuvelier, and species of microgreens, expressed as FW or DW per cup (Fig. 1A), are due Berset (Brand-Williams et al., 1995). The analysis was done following to their specific physiological characteristics, the different cultivation the method of (Villalva et al., 2020) with some modifications. Briefly, time of each species and/or the density of seeds per cup. In general, the 2.925 mL of DPPH solution in methanol (0.1 mmol L− 1) was mixed with growth of the different biofortified microgreens did not change in terms 0.075 mL of methanolic extracts of microgreens, vortexed for 10 s, and of biomass produced compared to the control plants. This indicates that incubated in the dark for 30 min at room temperature. The DPPH biomass yield was not affected by the addition of Se. These results are remaining in the solution after this time was measured at 515 nm in the consistent with the fact that the plants showed no visible symptoms of 96-well microplate. Trolox standard solutions (0–1.25 mmol L− 1) were toxicity. Similar results were obtained for other short-term crops bio­ used to obtain the DPPH inhibition curve following the same treatment fortified with different Se concentrations, such as broccoli (Ramos et al., of the samples. The results were expressed as μmol of Trolox equivalent 2011), basil (Chomchan et al., 2017; Pannico et al., 2020) or rice antioxidant capacity (TEAC value) per gram of FW. (Puccinelli et al., 2019) where biomass yield was not affected by bio­ fortification. In the present work, we suggest that this effect is probably due to the fact that the plants are able to tolerate this Se concentration 2.6. Chlorophylls and carotenoids and eliminate the excess by transforming Se compounds into the volatile forms dimethyselenide and dimethydiselenide to avoid toxicity as Pigments were determined as total chlorophylls (T Chl) and total Chomchan et al. (2017) stated. These volatilized forms of Se are about carotenoids (T Car). Pigment extraction was performed by adding 10 mL 600 times less dangerous than inorganic Se. of acetone/water (80/20, v/v) to 100 mg of lyophilized and ground plant tissues. The mixture was stirred for 10 min, centrifuged at 3500 3.2. Total selenium, micro- and macronutrients concentration rpm for 10 min, and the supernatant was filtrated through a 0.45 μm syringe filter. The solution was made up to 25 mL with the acetone so­ The total selenium concentration, expressed on a DW basis, was lution. The absorbance of the solution was analyzed at 440, 646 and 663 significantly higher in the Se-enriched microgreens compared to their nm wavelengths (UV double-beam spectrophotometer from UNICAM, respective controls (Fig. 1B). Similar Se uptake was observed in kale model UV-2 200, USA). Pigment concentration was determined using (133 mg Se⋅kg− 1 DW) and kohlrabi (137 mg Se⋅kg− 1 DW) microgreens the equations for chlorophylls (equations (1)–(3)) of Lichtenthaler and on average, while it was significantly lower for wheat (28 mg Se⋅kg− 1 Welburn (Lichtenthaler and Wellburn, 1983) and the equation for ca­ DW). rotenoids (equation (4)) of Holm (1954), where A is the absorbance of The results obtained demonstrate the effectiveness of the experiment the samples at the corresponding wavelengths. The results were performed in the bioaccumulation of Se, achieving the biofortification of expressed as mg pigment 100 g DW− 1. these microgreens in accordance with previous works. Sprouts of green cabbage, kale and other Brassica species (Ávila et al., 2014) showed a ChlA (μg⋅mL− 1) = 12.21 x A663 – 2.81 x A646 (1) similar accumulation of Se (approximately 160 mg kg− 1 DW) when − 1 ChlB (μg⋅mL ) = 20.13 x A646 – 2.81 x A663 (2) treated with 50 μmol L− 1 of sodium selenate. In our findings, kale and kohlrabi microgreens accumulate more Se than wheat, confirming the − 1 TChl (μg⋅mL ) = ChlA + ChlB (3) known ability of Brassica species to accumulate large amounts of S (Table 2) and concomitantly Se due to their chemical similarity in terms TCar (μg⋅mL− 1) = 4.69 x A440 – 0.268 x TChl (4) of properties. The uptake, translocation, and metabolism of Se mimic those of S which plays the same role in biochemical systems. Therefore, the substitution of sulfur for selenium results in selenium-analogous compounds that increase the selenium content. 2.7. Total soluble proteins In another study (Islam et al., 2020), biofortification of wheat microgreens was performed under hydroponic conditions, but with Total soluble protein concentration was determined using the different selenite concentrations, a lower Se dose (12.7 μmol L− 1 Se), Bradford assay as described by D’Amato et al. (2018). Initially, 0.1 g of and higher total Se concentrations (140 mg kg− 1 DW) if compared to our dry shoots were mixed with 25 mL of 0.05 M Tris-HCl buffer (pH 9.0). experiment. The increase in Se accumulation could be related to the After centrifugation at 12,000 g for 20 min at 4 ◦ C, the supernatant absence of competition between Se and essential ion transporters in a containing the extracted soluble proteins was collected. To quantify the nutrient-deficient growth medium. In the present work, additional nu­ protein concentration, 30 μL of the supernatant was mixed with 1500 μL trients from tap water (Table S1), especially phosphate and sulfate ions, of Bradford reagent and vortexed. After a 5-min incubation to allow could affect Se uptake by wheat roots (Nothstein et al., 2016). color development, absorbance was measured at 595 nm. Bovine serum For other microgreens or sprouts, the total Se concentration found is albumin (BSA) was used as a standard for calibration, and protein directly related to the ability of the plants species to uptake and accu­ concentration in the sample was expressed as mg of BSA per gram of dry mulate Se in their shoots, the Se species used in biofortification (selenite weight (mg BSA⋅g− 1 DW). or selenate), the number of days of exposure to the treatment and 3 M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 Fig. 1. (A) Fresh weight (FW -green bars) and dry weight (DW- grey bars) and (B) Total Se concentration obtained for kale, kohlrabi and wheat microgreens treated with selenium (Se) and their respective controls. Bars indicate the mean ± standard deviation (SD) of three replicates from two different experiments (n = 6). Tukey’s significance at p ≤ 0.05 among treatments is indicated by different letters within the species of microgreens. Microgreen seeds and roots take up Se by passive diffusion using Table 2 sulfate or phosphate carriers and there is a clear difference in the uptake Macro (g⋅kg− 1 DW) and micronutrients (mg⋅kg− 1 DW) concentration in bio­ of selenite or selenate. Selenate is actively taken up by the roots via fortified kale, kohlrabi and wheat microgreens and the corresponding controls. sulfate channels (SULTRs) in their plasma membrane. Therefore, there is Macronutrients Mg P S K Ca competition with sulfate, with high concentrations of S suppressing (g⋅kg− 1 DW) selenate uptake and low levels enhancing it. Selenite uptake is driven by Kale 5.7 ± 10 ± 1 a 21 ± 2 a 23 ± 3 a 11 ± 2 other processes (phosphate transporters and aquaporins), as excess or 0.5 b b deficiency of P effectively alters the uptake of Se and aquaporins Kale + Se 5.9 ± 10.7 ± 23 ± 1 a 22 ± 4 a 12 ± 1 0.3 b 0.9 a b involved in silicon transport. Furthermore, in addition to the presence of Kohlrabi 7.6 ± 10.9 ± 22 ± 1 a 25 ± 4 a 19 ± 3 competing S and P, Se uptake rate also depends on the concentration of 0.7 a 0.6 a a plant-available Se in the soil and conditions in the rhizosphere such as Kohlrabi + Se 7.4 ± 11.0 ± 24 ± 3 a 26 ± 4 a 19 ± 3 pH and redox potential, as Se ions are absorbed through the surface of 0.3 a 0.6 a a Wheat 3.2 ± 8.7 ± 3.77 ± 12.3 ± 4.1 ± the plasma membrane via an electrochemical gradient into the 0.1 c 0.1 b 0.05 b 0.4 b 0.2 c epidermal cells of the roots (Trippe and Pilon-Smits, 2021) . Wheat + Se 3.1 ± 9.1 ± 4.0 ± 12.7 ± 3.8 ± Selenite is easily assimilated to organic forms in plant roots, limiting 0.4 c 0.5 b 0.3 b 0.7 b 0.6 c root-to-shoot translocation, while selenate is rapidly translocated. The Micronutrients B Mn Fe Ni Cu Zn Mo presence of nutrients such as S has a protective effect against Se toxicity (mg⋅kg− 1 DW) both through anionic competition during uptake in the roots and during Kale 52 43 ± 93 ± 1.7 13 ± 60 0.63 subsequent transport of Se in the plant and in the incorporation of Se ±8 9 ab 15 ab ± 2a ±6 ± versus S into proteins (Zhou et al., 2020). a 0.1 ab 0.09 b The macro- and microelement profiles obtained (Table 2) are com­ ab parable to those reported in previous works on microgreens or more Kale + Se 51 48 ± 108 2.1 10 ± 63 0.65 ±6 6a ± 15 ± 2b ±5 ± mature plants, however the available data published on the mineral a a 0.5 a 0.08 b composition of microgreens are scarce, limiting the comparison (Lenzi ab et al., 2019; Mezeyová et al., 2022; Newman et al., 2021; Pinto et al., Kohlrabi 56 38 ± 77 ± 1.6 11 ± 56 0.67 2015). ±7 4b 7b ± 2 ab ±3 ± a 0.1 b ab 0.09 b In the case of macronutrients (Mg, P, S, K and Ca), the most abundant Kohlrabi + Se 57 39 ± 82 ± 2.2 7±1 52 0.58 b element was K followed by S, Ca, P, and Mg of the three species tested, ± 4b 10 b ± c ±5 ± the two Brassicaceae microgreens (kale and kohlrabi) had similar 10a 0.2 a b 0.09 b amounts of K, S, and P, but differed in Ca and Mg levels, with kohlrabi Wheat 58 52.8 79 ± 1.8 13.2 36 1.36 having the highest values. Wheat had the lowest macronutrient levels ±6 ± 0.2 6b ± ± 0.6 ±1 ± 0.04 a a 0.2 a c a among microgreens. No significant differences were found between the ab microgreens biofortified with Se and their respective control in terms of Wheat + Se 42 48 ± 80 ± 1.6 14 ± 44 1.50 the concentration of the above-mentioned elements. ±4 4a 10 b ± 2a ±8 ± 0.20 Among them, it should be highlighted that the S concentration in the a 0.2 bc a ab three crops studied was not affected by Se biofortification, considering that the preferential uptake of selenate or sulfate differs among plant Values are expressed as the mean ± SD of 3 replicates from two different ex­ species. The similarity between these two elements can lead to the periments (n = 6) of each microgreen. Tukey’s significance at p ≤ 0.05 between substitution of S-amino acids by Se-amino acids, which results in treatments is indicated by different letters for each nutrient. negative changes in the tertiary structure of the protein and leads to Se toxicity for the plants (Gupta and Gupta, 2017). presence of nutrients in the medium (D’Amato et al., 2018; Newman In contrast, significant differences were observed between micro­ et al., 2021; Puccinelli et al., 2019). Plants species differ strongly in the greens, for micronutrients, except for B. As expected, Fe was the uptake and accumulation of Se in shoots and in their ability to tolerate microelement with the highest accumulation in microgreens, followed high concentrations of Se in the roots and/or shoots. 4 M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 by Zn, B, and Mn with values close to half and similar ranges between conducted by (Tomas et al., 2021), kale and kohlrabi showed similar them. The micronutrients with the lowest values and in accordance with trends in antioxidant activity, but the values largely depended on the the need that plants have for them were Cu, Ni and Mo. The highest antioxidant activity assay and the cultivation parameters of the concentrations were found in kale (Fe, Mn, and Zn) and wheat (Cu and microgreens. Mo) compared to kohlrabi, contrary to the values of the macronutrients. Considering that the accumulation of polyphenols in plants is usually Statistically significant differences between the control and Se-enriched associated with stress conditions during the growth cycle (Gupta and microgreens, were found only for Cu for kale and kohlrabi (Cu con­ Gupta, 2017) , such as the presence of Se in excessive doses, our results centration decreases with biofortification) and Ni for kohlrabi (Ni con­ suggest that the Se concentration of 20 μmol L− 1 used for biofortification centration increases with Se uptake). Some works report the influence of does not induce abiotic stress in microgreens species. Other studies have Se on the transport and accumulation of micronutrients such as Cu or Ni reported comparable results when similar concentrations of Se were (Cipriano et al., 2022) as Se taken up by plants can alter the ionic used in different microgreens species (Newman et al., 2021; Puccinelli permeability coefficient in the cells of the plasma membrane, and thus et al., 2021). Due to its antioxidant effect, Se counteracts oxidative stress the uptake of other ions such as micronutrients. Pazurkiewicz-Kocot at low concentrations by inhibiting lipid peroxidation and increasing the et al. (2008) reported that Se reduces Cu concentration in the leaves activity of glutathione peroxidase (GSH-Px) with a positive effect on of biofortified corn and suggested that these may be the first symptoms plant growth at low concentrations. Increased antioxidation associated of the effects of Se on plants. Other possible explanations for reduced with an increase in GSH-Px activity can delay plant senescence and nutrient uptake are reduced root growth and poor root penetration decrease postharvest losses. On the contrary, at high concentrations of under Se stress or competition between Se and nutrient transporters and Se, particularly selenite, it can function as a pro-oxidant, causing bioligand sites, that inhibit the transfer of nutrient elements (Qu et al., oxidative stress in leafy vegetables, by increasing H2O2, malondialde­ 2023). hyde (MDA) content and lipid peroxidation (Puccinelli et al., 2017). Therefore, it can be assumed that biofortification with Se (20 μmol Sometimes, when Se does not induce changes in total phenolic com­ L− 1) for does not affect the uptake and accumulation of macro- and pounds, its effect may be on a specific compound and a phenolic com­ micronutrients for these three plants species. These minerals are of pound profile must be performed. There is, however, disagreement in crucial importance for the nutraceutical properties and consumption of the literature about the impact of selenium (Se) on phenolic content and these plants as foods. antioxidant activity. These differences can be related to plant species, Another aspect to consider is the recommended daily allowance growth stage, and Se treatments. (RDA) of 55 μg Se⋅day− 1 for adults to maintain the proper functioning of metabolism and the expression of selenoproteins (Institute of Medicine 3.4. Pigments: chlorophylls and carotenoids and Food and Nutrition Board, 2000) The estimated dietary Se intake (EDI, μg⋅day− 1), from the consumption of Se-enriched kale, kohlrabi, The values of photosynthetic pigments are presented in Table 4. The and wheat microgreens, is shown in Table 3. These values were calcu­ concentration of chlorophyll A was the highest in wheat microgreens lated on FW basis and assuming a daily consumption of 20 g of fresh (651 ± 92 mg⋅100 g− 1 DW) and similar in kale and kohlrabi. Chloro­ microgreens per person (Lenzi et al., 2019; Pinto et al., 2015). phyll B and total chlorophyll values had no significant differences be­ The values obtained indicate that the daily consumption of these tween the three species. The level of carotenoids was also similar plants could provide sufficient Se to meet the RDA criteria. In all cases, between species. These results suggest that the pigment content is var­ the results obtained are below the toxic threshold set at more than 400 iable between microgreens species, considering that the concentration μg⋅day− 1 (Institute of Medicine and Food and Nutrition Board, 2000). of these phytochemical compounds increases during leaf development. However, it should be noted that other foods naturally rich in Se present However, no significant changes were observed in the concentration in the diet can increase total Se intake. of Chls and Car between the biofortified microgreens and their respec­ tive controls, except for carotenoids in kohlrabi which increases from 96 to 130 mg⋅100 g− 1 DW with Se supply. 3.3. Bioactive compounds: total polyphenolic compounds and antioxidant Biofortification with Se could influence the biosynthesis of chloro­ activity phylls and carotenoids in microgreens by promotioning electron flow in the respiratory chain, protecting chloroplast enzymes and producing Se-biofortification of microgreens is not only suitable for supplying radical oxygen species (ROS), according to Lanza and Reis (2021). humans and animals with the necessary amounts of this nutrient, but However, our results indicate that, in general, there is no significant also for influencing the content of bioactive compounds produced by influence of Se in the accumulation of chlorophyll or carotenoid pig­ these plants, which have beneficial effects on health and metabolism ments at the biofortification levels achieved for these species. The cur­ (Pannico et al., 2020). rent findings are consistent with the concentration of bioactive In our study, the total polyphenolic compounds (TPC) content and compounds, as no indication of stress were observed. antioxidant activity of the three microgreens were not affected by Se The increased level of carotenoids in kohlrabi may be directly related treatment (Fig. 2). In contrast to antioxidant activity, no significant to biofortification. In previous works on Se biofortification in wheat differences were found in TPC between the species. The TEAC value was microgreens (Islam et al., 2020), the total carotenoid content increased higher in kohlrabi + Se (10.3 ± 0.6 μmol Trolox⋅g− 1 FW), followed by compared to control plants. The authors suggested that this could be kale + Se (8.3 ± 0.7 μmol Trolox⋅g− 1 FW) and the lowest value was related to the high rate of photosynthesis and the application of Se in the obtained in wheat + Se (7.3 ± 0.2 μmol Trolox⋅g− 1 FW). In a study restoration of chloroplasts damaged by environmental stress and ROS. In that work, biofortification was carried out with selenite in different Table 3 concentrations and growth periods compared to the present work, which Se concentration in FW and estimated daily intake (EDI) of Se from microgreens resulted in an increase in TPC and antioxidant activity, as well as pig­ consumption. ments, but in a decrease of plant performance. When these results are Microgreens Se concentration in FW (μg Estimated dietary intake of Se compared to our own, they confirm that many parameters can influence Se⋅g− 1 FW) (EDI, μg⋅day − 1) the biofortification process and that plants can produce different Kale + Se 8.6 172 amounts of phytochemical compounds at different Se concentrations. It Kohlrabi + 4.8 96 is important to note that the effect of photoperiod on chlorophylls levels Se can vary depending on plant species and environmental conditions. Wheat + Se 5.4 108 Other factors, such as temperature and nutrient availability, can also 5 M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 Fig. 2. Total polyphenolic compounds (A) and antioxidant activity (B) of kale, kohlrabi, and wheat microgreens biofortified with Se and their corresponding controls. Values are expressed as the mean ± SD of 3 replicates from two different experiments (n = 6) for each microgreen. Tukey’s significance at p ≤ 0.05 between treatments is indicated by different letters. of proteins with increasing selenium levels may be explained with the Table 4 interference of Se on sulfur assimilation and thus on the selenoamino Chlorophylls (ChlA, ChlB and TChl) and total carotenoids (TCar) concentrations of acids acid synthesis in these two cruciferous microgreens (D’Amato kale, kohlrabi, and wheat microgreens biofortified with Se and their corre­ sponding controls. et al., 2018) . The reasons for the differential response in wheat microgreens warrant further investigation. Microgreens ChlA (mg⋅100 ChlB (mg⋅100 TChl (mg⋅100 TCar (mg⋅100 Overall, the results of this analysis shed light on the complex rela­ g− 1 DW) g− 1 DW) g− 1 DW) g− 1 DW) tionship between Se biofortification, differences between microgreens Kale 456 ± 41 b 186 ± 15 a 642 ± 55 a 137 ± 9 ab or plants species, and total soluble protein concentration. Understanding Kale + Se 457 ± 56 b 181 ± 18 a 638 ± 73 a 133 ± 10 a Kohlrabi 394 ± 29 b 163 ± 12 a 557 ± 40 a 96 ± 12 c these variations is crucial to control the nutritional potential of micro­ Kohlrabi + 489 ± 76 bc 180 ± 17 a 669 ± 93 a 130 ± 23 ab greens and optimize their growth conditions to meet specific dietary Se requirements or nutritional preferences. Wheat 651 ± 92 a 167 ± 11 a 818 ± 93 a 136 ± 31 ab The most significant properties of each biofortified microgreen spe­ Wheat + Se 570 ± 58 ac 164 ± 10 a 734 ± 65 a 104 ± 20 bc cies are summarized in Fig. 4. Values are expressed as the mean ± SD of 3 replicates from two different ex­ periments (n = 6) for each microgreen. Tukey’s significance at p ≤ 0.05 between 4. Conclusions treatments is indicated by different letters. Selenium biofortification of kale, kohlrabi, and wheat microgreens affect chlorophyll production and accumulation in plants. Therefore, it seems to be a suitable alternative to increase the accumulation of this is necessary to consider all these factors when studying the relationship element in these highly nutritious crops using inorganic Se salts. High between photoperiod and chlorophyll levels in plants. levels of Se were obtained in all microgreens with this methodology, especially for the two Brassicaceae species. The incorporation of Se was achieved without affecting plant biomass, the level of essential macro- 3.5. Total soluble proteins and micronutrients, the production of bioactive compounds, antioxidant capacity, and pigment biosynthesis. Furthermore, for all three species, Total soluble protein (TSP) content was affected by selenium in kale Se intake is within the recommended limits in the human diet to achieve and kohlrabi microgreens but not in wheat specie (Fig. 3). The increase Fig. 3. Protein content in Se biofortified microgreens and their corresponding controls. Values are expressed as the mean ± SD of 3 replicates from two different experiments (n = 6) for each microgreen. Tukey’s significance at p ≤ Fig. 4. Summary of the most significant properties of the Se-biofortified 0.05 between treatments is indicated by different letters. microgreens. 6 M. Viltres-Portales et al. Plant Physiology and Biochemistry 206 (2024) 108283 the health benefits associated with Se. 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