For fresh samples of lettuce leaves and roots (directly after harvest), dry matter content was assayed at 105°C. The content of L-ascorbic acid in leaves was analyzed by capillary electrophoresis after the homogenisation of 20-g samples in 80 cm³ of 2% oxalate acid (puriss. p.a., Avantor Performance Materials) and further centrifugation for 15 min at 4,500 rpm, 5°C. The supernatants were filtered through a 0.25-μm cellulose acetate membrane filter and analyzed using a PA 800 Plus capillary electrophoresis system (Beckman Coulter, Indianapolis, IN, USA) with diode array detector (DAD) detection. Capillaries of 50 μm i.d. and 365 μm o.d. and those of a total length of 50 cm (40 cm to detector) were used. A negative power supply of 25 kV was applied. The running buffer solution was prepared as proposed by Zhao et al. (2011), containing 30 mM NaH₂PO₄ (puriss. p.a., Avantor Performance Materials), 15 mM Na₂B₄O₇ (puriss. p.a., Sigma–Aldrich) and 0.2 mM cetyltrimethylammonium bromide (CTAB) (puriss. p.a., Sigma–Aldrich) (pH 8.80).

In order to determine the content of sugars and phenolic compounds, fresh leaves were extracted with boiling 96% ethanol (Destylernia ‘Polmos’ Sp. z o.o., Kraków, Poland) using a reflux condenser. The levels of fructose, glucose and sucrose (and their sum as total sugars) were assessed by a PA 800 Plus capillary electrophoresis system (Beckman Coulter) with DAD detection. Capillaries of 25 μm and 365 μm o.d. and those of a total length of 60 cm (50 cm to detector) were used. A power supply of 30 kV normal polarity was applied. The detector was set at 205 nm. The running buffer solution contained 15 mL 36 mM Na₂HPO₄, 130 mM NaOH (puriss. p.a., Avantor Performance Materials) and 1.93 mM β-cyclodextrin (puriss. p.a., Sigma–Aldrich) (pH 12.7).

The content of phenols, phenylpropanoids, flavonols, and anthocyanins was determined spectrophotometrically after sample reactions with 0.1% HCl (puriss. p.a., Avantor Performance Materials) dissolved in ethanol (Fakumoto and Mazza, 2000).

Fresh lettuce roots and leaves (after being washed in distilled water) were dried at 70°C in a laboratory dryer with forced air circulation and ground in a FRITSCH Pulverisette 14 variable speed rotor mill (Idar-Oberstein, Germany) using 0.5-mm sieve. Samples were subsequently analyzed with respect to the contents of I, Se, P, K, Ca, Mg, S, Na, B, Cu, Fe, Mn, Mo, and Zn by the ICP-OES technique (using an ICP-OES Prodigy Spectrometer, Leeman Labs, New Hampshire, MA, USA); N by the Kjeldahl method; and selenomethionine (SeMet; Acros Organics, Geel, Belgium), selenocysteine (SeCys; Sigma–Aldrich), proline (Sigma–Aldrich) and SA (puriss. p.a., Avantor Performance Materials) using capillary electrophoresis via a PA 800 Plus system (Beckman Coulter).

Determination of I and Se content after tetramethylammonium hydroxide (TMAH) extraction: amounts of 0.5 g air-dried leaf or root samples, 10 cm³ double-distilled water and 1 cm³ of 25% TMAH (Sigma–Aldrich) were added to 30-cm³ Falcon tubes. After mixing, samples were incubated for 3 h at 90°C. After incubation, samples were cooled to a temperature of approximately 20°C and filled to 30 cm³ with double-distilled water. After mixing, samples were centrifuged for 15 min at 4,500 rpm. The measurements of I and Se content using an ICP-OES spectrometer (Leeman Labs) were conducted in the supernatant without decanting (PN-EN 15111, 2008; Smoleń et al., 2016).

Determination of SeMet, SeCys and proline content in lettuce: in 30-cm³ Falcon tubes, 5 cm³ of solution containing 40 mg protease and 20 mg lipase in demineralised water were added to 0.1-g air-dried plant samples (leaves or roots). Samples were incubated for 16 h at 20°C and then centrifuged for 15 min at 4,500 rpm (Zhao et al., 2011). The aliquots of 0.5 cm³ supernatants were transferred into 1.5-cm³ Eppendorf tubes and centrifuged for 10 min at 10,000 rpm. Measurements of SeMet, SeCys and proline content were conducted using the capillary electrophoresis technique with laser-induced fluorescence detection (PA 800 Plus, Beckman Coulter). The derivatisation procedure by Arlt et al. (2001) was slightly modified in order to prepare extracts for the laser-induced fluorescence measurements. One hundred microliters of plant extract and 100 μL carbonate buffer + fluorescein isothiocyanate mixture (4.9 mL carbonate buffer with final pH 9.2 in the mixture + 100 μL of 210-μM fluorescein isothiocyanate dissolved in acetone), after mixing, were incubated for 2 h in the dark. An amount of 0.2 M carbonate buffer at pH 11.2 was applied (Na₂CO₃, trace analysis quality; Sigma–Aldrich)_. The analogous derivatisation procedure was applied to standard reagents and blank samples (double-distilled water). SeMet, SeCys and proline were analyzed using capillaries of 50 μm i.d. and 365 μm o.d. and those of a total length of 50 cm (40 cm to the detection window). Separation was conducted for 60 min at 7 kV using the standard PA 800 Plus laser (Beckman Coulter) with 488-nm and 500-nm long-pass filters in the detector. The running buffer contained 45 mM β-cyclodextrin + 80 mM sodium borate (Sigma–Aldrich) (pH 9.2).

Determination of SA content: a plant sample amount of 0.2 g was placed in 7-cm³ polypropylene tubes; 5 cm³ of demineralised water was added and mixed for 1 min. Samples were incubated for 30 min in a 60°C water bath, cooled to room temperature and centrifuged for 15 min at 5°C at 4,500 rpm. Samples were then filtered through cellulose acetate membrane filters and analyzed by capillary electrophoresis via a PA 800 Plus system (Beckman Coulter). Capillaries of 75 μm i.d. and 365 μm o.d. and those of a total length of 30 cm (20 cm to the detector window) were used. Separation was conducted at -25 kV and the detection of SA at 205 nm. The running buffer solution contained 10 mM Tris (Sigma–Aldrich) at pH 2.78 set by formic acid (puriss. p.a., Avantor Performance Materials) (Coolen et al., 1998).

Determination of macro- and microelements: in order to analyze the total content of P, K, Ca, Mg, S, Na, B, Cu, Fe, Mn, Mo, and Zn using an ICP-OES spectrometer (Leeman Labs), plant samples were digested in 65% super pure HNO₃ (Merck, Whitehouse, Station, NJ, USA) in a CEM MARS-5 Xpress (CEM World Headquarters, Matthews, NC, USA) microwave digestion system (Pasławski and Migaszewski, 2006). Total N content of leaf and root samples was assayed by the Kjeldahl method with the use of a VELP Scientifica UDK 193 distillation unit (Usmate, Italy).

Samples from the third-youngest lettuce leaf were collected directly before harvest, during the 10th week of plant cultivation in the nutrient solutions with I, Se, and SA. Three biological samples were collected simultaneously from each experimental treatment. Samples were frozen in liquid N immediately after collection and stored at -80°C until use.

The Spectrum^TM Plant Total RNA Kit (Sigma–Aldrich, Cat # STRN250) was used for RNA extraction of leaves, and 1 μg RNA was subsequently treated with RNase-free DNase I (Ambion Inc., Austin, TX, USA, Cat # AM2222) and 20 U⋅μL^-1 RiboLock RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, USA, Cat # EO0381) according to the manufacturers’ instructions. RNA concentration was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 and 280 nm, and the 260/280 nm ratio within the range of 2.00–2.15 was retained. RNA integrity and quality were verified by electrophoresis on 1% denaturing agarose gel. First-strand cDNA synthesis was performed using an iScript cDNA Synthesis Kit (Bio-Rad laboratories, Hemel Hempstead, UK, Cat #1708891) in accordance with the manufacturer’s instructions. The final concentration and quality of cDNA were determined spectrophotometrically using a Nanodrop 2000c spectrophotometer (Thermo Scientific). The 10-fold dilution products of the generated cDNAs were used for qRT-PCR analyses.

Sequences encoding the SMT gene, available in the GenBank database¹, were aligned using ClustalW 2.1 software (Larkin et al., 2007). Primers were selected from conserved regions of the alignment, designated SMT-F (5′-ACACAGGAGTTGGGAATGAAG-3′) and SMT-R (5′-CTCTGATGGTGGTTGGTGTT-3′), generating a fragment of 108 bp. These sequences were submitted to BLAST of the National Center for Biotechnology Information to verify their analytical specificity, whereas the formation of dimers, hairpins and melting temperature was assessed with OligoAnalyzer 3.1 software². Transcript levels were normalized to the expression level of actin (GenBank Acc. No. AB359898.1), as determined using the primers LsACT-F (5′-AGGTGTCATGGTTGGCATGGGA-3′) and LsACT-R (5′-TGTTCTTCAGGGGCGACACG-3′). The sizes of the amplified fragments were confirmed by agarose gel electrophoresis.

In addition, amplification of the serine acetyl transferase (SAT) gene involved in the formation of O-acetyl-L-serine, which represents a regulatory step in selenoamino acid assimilation, was performed (Saito, 2004). Three SAT-specific primer sets developed by Ramos et al. (2011) were analyzed. Unfortunately, non-specific PCR products on the agarose gels were obtained, which were inappropriate for qRT-PCR analyses.

Real-time PCR reactions consisted of 12.5 μL Maxima SYBR Green/ROX qPCR Master Mix (2x) (Thermo Fisher Scientific, Cat # K0221), 0.7 μM of each primer, 2 μL of cDNA and up to 25 μL of nuclease-free, DEPC-treated water (diethylpyrocarbonate; Thermo Fisher Scientific, Loughborough, UK, Cat # BP561-1). PCR was performed using a StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in 96-well plates with the following cycle parameters: 95°C for 10 min; 40 cycles at 95°C for 15 s; and 52°C for 1 min, and a standard melting curve (95°C for 15 s, 60°C for 1 min and 95°C for 15 s with a reading at every 0.3°C) was used to assess the specificity of PCR according to the melting temperature profile of the reference strain. Each run included a negative control and a cDNA reaction without reverse transcriptase to rule out DNA contamination. Each PCR reaction was repeated three times; therefore, the final number of repetitions for each experimental variant was three technical instrumental replicates × 2 repeated experiments × 3 biological samples.

The ΔΔC_T method (Livak and Schmittgen, 2001) was used to normalize and calculate ratios of expression levels (relative fold changes) relative to a housekeeping gene (actin), whose expression did not change in response to changes in the composition of the nutrition solution.

The percentage of recommended daily allowance for I (RDA-I) and Se (RDA-Se) supplied from one serving of 50 g fresh lettuce leaves was calculated using the results of I and Se content in fresh lettuce leaves as well as the recommended daily intake of these two elements for adults: 150 μg I and 55 μg Se daily (Food and Nutrition Board – Institute of Medicine, 2000; Andersson et al., 2007).

Tested factors had a significant influence on the content of sugars (fructose, glucose, sucrose, and their sum) but did not affect the dry matter content (leaves and roots) and the content of ascorbic acid, phenolic compounds, phenylpropanoids, flavonols and anthocyanins in lettuce leaves (Tables 1 and 2).

In comparison to the control, all tested combinations with I + Se applied separately or together with SA contributed to a comparable increase in total content of sugars in leaves (Table 1). In comparison to the control, significant and comparable increases of fructose and glucose were noted in the combinations with I + Se, I + Se + 1.0 SA and I + Se + 10.0 SA. Only the introduction of I + Se or I + Se + 0.1 SA into the nutrient solution increased sucrose accumulation in leaves relative to the control. No tested combination decreased the level of sugars in lettuce plants.

A significant and diverse influence of tested factors on the content of macro- and micronutrients in lettuce was noted (Tables 3 and 4). The only exception was the lack of significant differences in Ca and Mo accumulation in lettuce among combinations.

In roots of plants from all combinations, a significant increase of P concentration was found with respect to the control. In addition, there was an increase of N and S content after the application of Se + 0.1 SA and I + Se + 1.0 SA as well as of K in the combinations with I + Se + SA at all SA doses. A negative effect of I + Se and I + Se + SA was mainly manifested by the decreased contents of Ca, Mg, Na (Table 3), B, Cu, Fe, Mn, Mo, and Zn (Table 4). In each case, lower levels of these nutrients in roots were revealed compared to the control. To the greatest extent, a decrease of the content of analyzed nutrients in lettuce roots was noted in the respective combinations: Ca, Cu, Fe, and Mn after the application of I + Se + 1.0 SA; Na after the application of I + Se + 0.1 SA; Zn after the application of I + Se + 10.0 SA; Mg comparably in the combinations with I + Se + 0.1 SA and I + Se + 1.0 SA; and Mo comparably in the combinations with I + Se + 0.1 SA and I + Se + 10.0 SA.

Introduction into the nutrient solution of I, Se, and SA improved the accumulation of Na in lettuce leaves compared to the control, but the increase did not vary between tested combinations (Table 3). In addition, the application of I + Se + 0.1 SA led to the increase of Cu, Fe, and Mn content in leaves (Table 4). In all remaining combinations, a decrease of Cu content in lettuce was noted, with no statistical significance for I + Se + 10.0 SA. In the case of Fe, a decrease in its concentration was found in leaves of plants grown in the presence of I + Se and I + Se + 1.0 SA. A tendency of diminishing N, P, K, Mg, S, B, and Zn contents in lettuce leaves was noted for all tested combinations compared to the control (Tables 3 and 4), but statistically significant changes were noted only for K, S, B, Cu, Fe, and Zn after the application of I + Se.

It should be noted that, compared to the control, all tested treatments led to simultaneous decreases in Mg contents in leaves and roots. In the case of Zn, only for the applications of I + Se + 1.0 SA and I + Se + 10.0 SA did its contents decrease in both above- and belowground parts of plants.

The increase in the content of I and Se in lettuce after the application of these two elements allowed for increasing the possibility of covering RDAs for both nutrients after the consumption of a single 50-g serving of lettuce leaves (Table 5). In comparison to the control, RDA-I values increased from 3.5% to 388.8–441.1% for I + Se + 0.1 SA and I + Se + 10.0 SA, respectively. In the case of RDA-Se, growth was less distinctive (i.e., from 5.6% to 39.1–53.7% for I + Se + 1.0 SA and I + Se, respectively).

Introduction of I, Se and SA into the nutrient solution widened the molar ratio of I and Se content in lettuce plants from 1.3:1 (control) to 12.4:1 (I + Se + 0.1 SA) and 17.0:1 (I + Se + 1.0 SA).

Leafy vegetables belong to a group of plants that accumulate substantial amounts of I (Dai et al., 2004; Hong et al., 2008). On the other hand, vegetables from this group (spinach, pak choi) exhibit higher sensitivity to I fertilization than, for example, carrot (Dai et al., 2004).

Some plant species, including those of the Asteraceae family with lettuce, are described as hyperaccumulators. They can accumulate Se up to 100 times more compared to other vegetable crops (Beath et al., 1939a,b). In hyperaccumulators, Se is mainly present in the methylated form (methyl-SeCys) but not SeMet and SeCys. The accumulation of SeMet and SeCys occurs in non-accumulators – those plants that take up small amounts of this element (Yuan et al., 2013). The process of SeCys transformation into methyl-SeCys (by the SMT enzyme) in the Se metabolism pathway plays a protective role for plants. It consists of the conversion of potentially toxic Se-amino acids into less harmful selenometabolites (Ellis et al., 2004).

According to Winkel et al. (2015), the process of SeCys conversion to methyl-SeCys is one of the four independent metabolic pathways responsible for the synthesis of various selenoorganic compounds from SeCys. With the participation of L-cysteine oxidase, methyl-SeCys undergoes transformation into methylselenocysteine selenoxide (MSeCysSeO), which is further converted by cysteine sulphoxide lyase to volatile DMDSe. DMDSe also can be formed in another pathway converting SeMet into Se-methylseleno-L-cysteine. Se-Met alone is synthesized through conversion of SeCys by a few steps.

Another volatile Se compound that is formed through methylation and undergoes volatilisation is DMSe, produced in a separate few-step metabolic pathway from SeMet. Volatilisation of DMDSe and DMSe contributes to a decrease of Se content in plant leaves (Winkel et al., 2015).

In the combination with I + Se application (compared to the control), SMT gene expression in lettuce substantially increased, which seems justified by the content of total Se and SeMet. Interpretation of the results is, however, far more complex (Table 6). Aside from the analyzed SMT gene expression, chemical analyses of leaves and roots clearly indicate that, depending on the dose, exogenous SA (applied together with I + Se) has a significant and diverse effect on plants, mostly with respect to the syntheses of SeMet, SeCys and proline in leaves and roots. In addition, significant differences were observed in SA accumulation and the functioning of mineral nutrition, including the uptake and accumulations of I and Se as well as macro- and micronutrients in lettuce leaves and roots.

It needs to be mentioned that I also undergoes methylation in plants, forming volatile CH₃I, a process regulated by S-adenosylmethionine-dependent halide/thiol methyltransferase (Itoh et al., 2009). Aside from this, plants have the ability to conduct SA methylation into MeSA via SA carboxyl methyltransferase (Tieman et al., 2010). All of these processes (syntheses of DMDSe, DMSe, CH₃I and MeSA) require an energy input. In the available literature, there is no information on whether a common mechanism regulating these processes occurs or whether they act independently.

In studies conducted by Smoleń et al. (2014), the application of I and/or Se (alone or simultaneously) to the nutrient solution had no effect on the contents of N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Mo, and Zn in leaves and roots of lettuce cultivated in an NFT system. Importantly, introduction into the nutrient solution of KIO₃ + Na₂SeO₄ (1 mg I + 0.5 mg Se⋅dm^-3 and 1 mg I + 1.5 mg Se⋅dm^-3) did not affect the contents of macro- and micronutrients in lettuce leaves and roots compared to the control. The causes of obtaining adverse results compared to those presented by Smoleń et al. (2014) might include the use of another lettuce cultivar and application of the chemical form of Se – Na₂SeO₃ instead of Na₂SeO₄ (Smoleń et al., 2014). In both studies, I was applied in the iodate form (KIO₃). When plants were cultivated in hydroponics, diverse actions of Se(IV) and Se(VI) on lettuce (Ríos et al., 2008, 2010) and cucumber were noted (Hawrylak-Nowak et al., 2015). Blasco et al. (2012) revealed a diverse influence of I^- and IO3− introduced through the nutrient solution on the mineral composition of lettuce plants. This effect was strongly dependent on the dose, including a substantial decrease of N, P, and K content in lettuce below the optimal range for this species after the application of 80 μM I^-. Conversely, introduction of IO3− affected the mineral nutrition of lettuce plants to a lesser extent, even improving the levels of Mg and Fe in plants. It needs to be mentioned that in the studies by Smoleń et al. (2014), simultaneous application of KIO₃ + Na₂SeO₄ had no effect on the uptake and accumulation of I and Se (in roots and leaves) compared to plants grown in the nutrient solutions containing I or Se separately.

Blasco et al. (2012) presented results concerning macro- and micronutrient sufficiency intervals in mature leaves of lettuce plants. A thorough analysis of these data indicates that the content of macro- and micronutrients in plants from the present study stayed within the optimal range for lettuce. In the case of K, its content in leaves was even supra-optimal. This could indicate that tested doses of I, Se, and SA did not exceed the tolerance level for lettuce plants (no decrease of plant biomass) while substantially modifying the mineral nutrition (in both leaves and roots).

Smoleń et al. (2015) revealed that introduction into the nutrient solution of KI + SA and KIO₃ + SA significantly improved I accumulation in tomato plants (in leaves and fruits) compared to plants grown in nutrient solution containing only KI or KIO₃. Importantly, the increase of I content in tomato leaves and fruits after the application of KIO₃ + SA was a few dozen times higher than that after the application of KI + SA. Smoleń et al. (2014, 2015) did not document molecular or biochemical mechanisms regulating the uptake and accumulation of I, Se or macro- and micronutrients and, in the case of tomato, also after the exogenous application of SA (Smoleń et al., 2015).

In our opinion, results of the present study do not provide a clear interaction at the biochemical and molecular levels among the metabolic pathways of I, Se, and SA. The results can only describe to what extent additional application of SA modifies the effect of simultaneous application of I + Se on plants.

Proline is an amino acid synthesized by plants in response to biotic or abiotic stresses (Hayat et al., 2012). In turn, SA stimulates plant reaction/resistance mechanisms to both types of stress factors (Hayat et al., 2012). Despite the lack of visual symptoms of toxic effects of tested compounds on plants, the highest concentration of proline was noted in leaves and roots of lettuce grown in the nutrient solution containing I + Se + 0.1 SA. This might have resulted from the greatest accumulation of I, Se, SeMet, Cu, Fe, and Mn in leaves and SeCys in roots of these plants. A lower expression of the SMT gene also was observed in plants of that treatment compared to plants that received the application of I + Se. Therefore, SA application at the lowest dose (0.1 mg SA⋅dm^-3) may have limited Se methylation and volatilisation in the form of DMDSe by SMT enzymatic activity. The whole assessment of the conducted molecular and chemical analyses does not provide justification for the hypothesis that the dose of 0.1 mg SA⋅dm^-3 was a stress factor to plants. This approach is further confirmed by the lack of differentiation in the content of phenolic compounds, the synthesis of which is also triggered by stresses (Ríos et al., 2008). Two hypotheses can be stated that explain the obtained results. The first one indicates that the decrease in SMT gene expression (combination of I + Se + 0.1 SA versus I + Se) could have resulted indirectly from the stimulation, through 0.1 mg SA⋅dm^-3 application, of more intensive catabolism (Zhang et al., 2013) or from SA methylation processes (Tieman et al., 2010). The other hypothesis includes the limitation of de novo synthesis of endogenous SA (Hayat et al., 2010) in lettuce leaves and roots after the application of 0.1 mg SA⋅dm^-3. In our opinion, introduction of exogenous SA, even at such a low dose, could have been sufficient to initiate the processes suggested in both hypotheses. As an effect, SA applied at the doses of 0.1 and 1.0 mg⋅dm^-3 could have been a key factor limiting the expression of SMT (I + Se versus I + Se + 0.1 SA as well as I + Se versus I + Se + 1.0 SA).

In this aspect, surprising was a significant increase in SMT expression (the highest relative expression ratio value) after application of the highest dose of SA (I + Se + 10.0 SA). In this combination, the tendency of decreasing (compared to I + Se + 0.1 SA) total contents of Se in leaves and SeCys in roots indicates that a 10.0-mg SA⋅dm^-3 dose of SA could have improved the methylation and volatilisation of DMDSe and/or DMSe. Lack of a negative influence of simultaneous application of I + Se + 10.0 SA on the sugar content in leaves indirectly suggests that photosynthesis was unaffected by tested factors and that it provided sufficient amounts of metabolites to cover the energy requirements for the mentioned processes.

Selenocysteine methyltransferase plays an important role in plant Se metabolism (Malagoli et al., 2015). Lyi et al. (2007) showed that SMT expression was dramatically up-regulated in broccoli plants exposed to selenate but was low in plants supplied with selenite. Furthermore, it needs to be mentioned that diverse plant species may exhibit different mechanisms of regulating SMT gene expression after the application of Se(IV) (i.e., SeO32− ion). In Se(IV)-tolerant mutants of Arabidopsis thaliana, after application of exogenous SeO32− , expression of the SMT gene is up-regulated. In plants sensitive to Se(IV), SeO32− decreases the expression of that gene (Zhang et al., 2006). The results of our study indirectly indicate that expression of the SMT gene in lettuce additionally can be regulated by exo-/endogenous SA or by its metabolic processes in plants.

Molecular aspects of Se, I and SA metabolism should become the goal of future studies to point out the enzyme-encoding genes, whose expression is modified by the tested factors, leading to increased accumulation of certain metabolites, whose synthesis is regulated by these enzymes.

Studies concerning lettuce biofortification with Se and I should be directed to integrate analyses at the genomic, transcriptomic, proteomic and metabolomic levels. Multi-omic approaches would allow for the characterisation of individual metabolic pathways and their interrelationships. A substantial difficulty in the identification and analysis of genes related to I and Se metabolism is, however, due to the fact that the lettuce genome is not fully known.

The optimal molar ratio of I:Se in daily food intake stays within 4.4–8.8:1, depending on age and gender. The RDAs for these elements are 150 μg I and 55 μg Se for adults and 200–300 μg I and 60–70 μg Se for pregnant and lactating women (Food and Nutrition Board – Institute of Medicine, 2000; Andersson et al., 2007).

A substantial but supra-optimal effect of lettuce biofortification with I and Se was obtained with respect to the possibility of balancing the diet concerning I and Se RDAs. In the case of I, the consumption of 50 g fresh leaves of lettuce would supply approximately four times more I than required. In the case of Se, 50 g lettuce leaves would supply 40–50% of recommended Se intake. In our consideration, the level of lettuce enrichment of both elements can be considered too high, as it is required to balance the entire daily intake with respect to the content of I and Se.

Regarding mild deficiencies of I and Se in the population, biofortification of lettuce or other leafy vegetables (and also other crop species) with I and Se should be conducted to cover the RDA percentage for I and Se (supplied with one serving) to approximately 10–30%. This results from the need to fully balance the diet with I and Se. For severe deficiencies of both elements, the applied doses of I and Se need to be higher, so the biofortified plants should become a substantial source of these micronutrients. It seems crucial to aim to achieve the optimal molar I:Se ratio in biofortified plants. With respect to the biofortification effect, it should not contribute to a decrease in the content of other mineral nutrients in plants, particularly Fe and Zn, whose deficiencies are a widespread problem throughout the world (White and Broadley, 2009). In our studies, plant biofortification with I and Se (without SA application) negatively affected the content of K, S, B, Cu, Fe, and Zn in lettuce plants, which would result in their lower introduction to the human diet.