Four different experiments were conducted in a glasshouse in Pisa (Tuscany, central-western Italy), with seedlings of two cultivars of sweet basil, “Tigullio” and “Red Rubin.” Seeds were purchased from Franchi Sementi (Milano, Italy). The seedlings were grown in hydroponic culture (floating system) in late spring or in early summer (see Table 1 for details). Inside the greenhouse, plants were grown under natural light.

The seeds of basil were sown using trays with rockwool plugs (Grodan Plug^®, Grodan Rockwool B.V., Roermond, The Netherlands). After sowing, the trays were maintained five days in a germination chamber with a constant temperature of 25°C and 75% relative humidity. Afterwards, they were moved to the greenhouse for the correct development of the plantlets, which were transferred in the hydroponic system at the stage of four visible true-leaves. In Experiments 1, 2, 3, each hydroponic system consisted of a polystyrene tray floating in a 50-L plastic tank (each tank hosted 16 plants, 8 “Tigullio” plus 8 “Red Rubin”), while 3-liter plastic pots were used in Experiment 4, each one hosting 4 plants of the same variety. Planting density was approximately 96 plants m⁻² of ground area. In all experiments, the nutrient solutions were prepared using tap water and appropriate amounts of salts of analytical grade (Carlo Erba Reagents, Milano, Italy) to avoid iodine contamination. The tap water contained 4.0 mM HCO₃-, 4.5 mM Cl, 0.25 mM S–H₂SO₄, 2.7 mM Ca, 0.8 mM Mg, 2.0 mM Na and negligible concentrations of other nutritive ions, including iodine (0.02 ± 0.01 µM I). The nutrient solution had a pH and an electrical conductivity (EC) of 5.5 and 2.4 dS m⁻¹, respectively, and contained the following concentrations of macroelements and trace elements: 10.0 mM N–NO₃-; 1.0 mM P–H₃PO₄; 3.8 mM S–H₂SO₄, 8.5 mM K; 4.5 mM Ca; 2.0 mM Mg; 2.0 mM Na; 4.5 mM Cl; 40.0 µM Fe; 25.0 µM B; 10.0 µM Mn; 10.0 µM Zn; 3.0 µM Cu; 1 µM Mo. The tested iodine concentrations ranged from 0.10 ± 0.03 µM (control) and 200 µM (Table 1). In each hydroponic system, the large volume of the nutrient solution limited the variations of pH (5.5–6.0), EC (2.3–2.5 dS m⁻¹) and ion concentrations, which were checked in all treatments every 2–3 days and remained within 3–5% the initial values throughout the experiments (data not shown).

The treatments were defined by a combination of two factors: the concentration of iodine in the nutrient solution and the cultivar (“Tigullio” and “Red Rubin”). In all experiments, the treatments were arranged in a totally randomized design with three replicates, each consisting of a tank or a pot.

The experimental treatments were differentiated one week after transplanting in the hydroponic system by adding potassium iodide (KI) or potassium iodate (KIO₃) of high purity (purity > 99%; Duchefa Biochemie B.V., Haarlem, The Netherlands) directly to the nutrient solution.

Four experiments were carried out with both basil genotypes.

In Experiment 1 we investigated the effect of iodine source (KI or KIO₃) and concentration (0.1, 10 and 100 µM) on plant growth. The experiment was conducted in summer 2015 and lasted 21 days.

In Experiment 2 KI was tested at the following concentrations: 0.1, 10, 50, 100, and 200 µM. The experiment was conducted in summer 2015 and lasted 16 days.

In Experiment 3 we studied the effect of the iodine source (KI or KIO₃) and its concentration in the nutrient solution on plant growth and leaf nutrient content. The following concentrations were used: 0.1, 50, 100, 200 µM for KI, and 100, 200, 400 µM for KIO₃. The experiment was conducted in spring 2016 and lasted 14 days.

In Experiment 4 we assessed the effect of 100 and 200 µM KI or KIO₃ on gas exchange at the two leaves of the 2^nd node. The antioxidant capacity and the contents of chlorophylls, carotenoids and total phenols were also assessed in the same leaves, along with the photosynthetic activity (through leaf gas exchange measurements). The experiment was carried out for 14 days in spring 2016.

In all the experiments, the symptoms of iodine toxicity appeared within three days of exposure as a reduction of stem elongation, leaf expansion and root development, and within one week as chlorotic interveinal yellow patches of the older leaves (Figure 1). The severity of symptoms increased with time, depending on the form and concentration of the iodine applied as well as on the cultivar. In KI-treated plants (Figure 1), early phytotoxicity symptoms appeared already after a few days as shorten new developed nodes, discolorations and chlorotic areas in the leaves. After 2 weeks iodine treatment, the plants showed serious phytotoxicity symptoms on all the leaves and few bottom leaves had also dropped.

The symptoms were less severe in “Red Rubin” than in “Tigullio” and when the plants were treated with KIO₃ than with KI at the same concentrations (Figure 2).

The green-leaved cultivar “Tigullio” grew faster than “Red Rubin” and after 21 days the total plant dry weight of “Tigullio” plants was approximately three times higher than the dry weight of “Red Rubin” plants.

Neither 10 nor 100 µM KIO3 concentrations affected plant growth in both cultivars. On the contrary, 100 µM KI in the nutrient solution significantly reduced the dry weight of roots, leaves and stems, respectively, by 22, 67, and 80% in “Tigullio,” and by 17, 22, and 23% in “Red Rubin.” When the plants were exposed to 100 µM KI, plant height and leaf area were reduced, respectively, by 64 and 68% in “Tigullio” and by 33 and 13% in “Red Rubin” (Table SM1).

After 16 days treatment, a reduction in plant growth was observed in both basil cultivars at KI concentrations higher than 10 µM; however, growth inhibition was less severe in “Red Rubin” than in “Tigullio” (Table 2). For example, compared to the control, 200 µM KI concentration reduced total plant dry matter and plant height, respectively, by 42 and 46% in “Red Rubin,” and both by 73% in “Tigullio”. In both cultivars, leaf iodine content increased with increasing KI concentration in the nutrient solution (Table 2). Iodine accumulation in leaf tissues was more pronounced in “Red Rubin” than in “Tigullio”: at 200 µM KI concentration, leaf iodine contents were 6,955 ± 915 in “Red Rubin” and 5,225 ± 380 mg kg⁻¹ DW in “Tigullio.”

The Maas-Hoffman equations for relative plant height or dry biomass against the KI concentration in the nutrient solution are reported in Table 3 and Figure SM1. In “Tigullio” the threshold iodine concentrations (t) for leaf DW, total plant DW and plant height were within 17.8 µM and 12.4 µM (on average, t was 16.0 µM), while the slope s was similar for the three growing parameters under consideration (on average, s was −0.42% µM⁻¹). Compared with “Tigullio,” “Red Rubin” showed a higher t (on average, it was 29.8 µM) and a lower absolute value of s (−0.24% µM⁻¹, on average).

When plant growth response was analysed against leaf iodine concentrations, t and s were, respectively, 272 mg kg⁻¹ DW and −0.015% mg⁻¹ kg DW in “Tigullio,” and 667 mg kg⁻¹ DW and −0.005% mg⁻¹ kg DW in “Red Rubin” (Table 3).

The main goal of this experiment was to determine if the growth inhibition of sweet basil due to toxic iodine concentrations in the nutrient solution was associated with a reduced uptake of other nutrients. Different concentrations of KI (50, 100, and 200 µM) or KIO₃ (100, 200, and 400 µM) were tested in order to induce different levels of growth inhibition.

As found in Experiments 1 and 2, “Red Rubin” showed a higher iodine tolerance than “Tigullio” and KI was more toxic than KIO₃ (Supplementary Material, Table SM2). The growth reduction induced by 400 µM KIO₃ was similar to the effect produced by the 50 µM KI treatment (Figure 2; Supplementary Material, Table SM2).

In the control, there was no important difference between “Tigullio” and “Red Rubin” as regards the leaf concentration of macronutrients and micronutrients, apart from a slightly lower content of P in “Red Rubin” (Table 4).

Iodine level did not influence the leaf concentrations of Mg (0.41% ± 0.04 mg kg⁻¹ DW), Mn (77 ± 12 mg kg⁻¹ DW) and Cu (14.1 ± 2.5 mg kg⁻¹ DW). Compared with the control, the 200 µM KI treatment decreased the leaf content of total N, P, K, and Zn, and increased the leaf content of Ca and Fe in both basil cultivars (Table 4). Evident effects of KIO₃ on leaf nutrient content were observed only in the 400 µM treatment in both cultivars, and consisted in a significant increase in the leaf content of Ca, Fe and Zn, without any significant reduction in the content of other nutrients.

The addition of iodine to the nutrient solution did not have important effects of leaf nitrate content (expressed on a fresh weight basis). Compared to the control (1,196 ± 85 mg NO₃⁻ kg⁻¹ FW) a significant increase in leaf nitrate content was observed only in “Tigullio” plants grown at 200 µM KI (1,661 ± 65 mg NO₃⁻ kg⁻¹ FW). In general, “Red Rubin” accumulated more nitrates in leaf tissues than “Tigullio.”

At the end of the experiment, in both the cultivars and for all the concentrations tested, KI produced a greater increase in iodine leaf content than the same concentration of KIO₃ (Figure 3). For example, a 14-days cultivation in a nutrient solution containing 200 µM KI or KIO₃ produced an iodine leaf content of 4,250 ± 460 or 687 ± 84 mg kg⁻¹ DW in “Tigullio” and 5,433 ± 218 or 717 ± 68 mg kg⁻¹ DW in “Red Rubin,” respectively. A significant linear relationship between iodine leaf content and KI or KIO₃ concentration in the nutrient solution was found for both cultivars. However, for the KIO₃ treatments, the slopes and intercepts of the regression lines for “Tigullio” and ‘Red Rubin’ were not significantly different (P < 0.05); therefore, the data of both cultivars were pooled and a single regression line was obtained (Figure 3).

In this experiment we investigated the relationship between CO₂ assimilation (A) and iodine content in plants of both cultivars grown with different concentrations of KI (50, 100 and 200 µM) or KIO₃ (100, 200 and 400 µM). The determinations were performed after 12 days of treatment on basal leaves (2^nd node) that were already present at the start of the experiment.

Both 100 and 200 µM KI caused a significant decrease of dry biomass accumulation and leaf area, while only 200 µM KIO₃ induced a slight reduction of these parameters (data not shown) in agreement with the results of the previous experiments. For the sake of brevity, only the mean values of the total leaf area at the end of the experiment are here reported, for “Tigullio” and “Red Rubin”: 906 ± 64 and 602 ± 55, 897 ± 50 and 564 ± 48, 824 ± 42 and 445 ± 37, 499 ± 51 and 426 ± 29, 376 ± 32 and 401 ± 21 cm² plant⁻¹ for control, 100 µM KIO₃, 200 µM KIO₃, 100 µM KI, and 200 µM KI treatments, respectively.

In the control, net photosynthesis (A) was approximately 50% lower in “Red Rubin” than in “Tigullio” (Figure 4A). In both basil cultivars, all tested KI and KIO₃ concentrations significantly decreased leaf photosynthesis (Figure 4A) while stomatal conductance (Gs) was significantly reduced only by KI (Figure 4B). In “Tigullio” plants, no significant effects of iodine were found on internal CO₂ concentrations (Ci; Figure 4C).

In general, the basal leaves of “Red Rubin” had a higher iodine content than those of “Tigullio” (Figure 4D). For example, at 200 µM KI leaf iodine content was 5,623 ± 995 mg kg⁻¹ DW in “Tigullio” and 7,720 ± 346 mg kg⁻¹ DW in “Red Rubin” (Figure 4D).

In both basil cultivars, a close negative correlation was found between A and iodine content as measured on the same leaves, with significant differences between “Tigullio” and “Red Rubin” as regards the intercept and the slope of the linear regression (Figure 5).

The content of carotenoids, total chlorophylls and phenols (gallic acid equivalents), and total antioxidant capacity were also determined in the same leaves sampled for leaf gas exchange measurements (Figure 6). In “Tigullio” a reduced content of total chlorophylls and carotenoids compared with the control (1.23 ± 0.04 and 0.26 ± 0.01 mg g⁻¹ FW, respectively) was observed with 100 and 200 µM KI (on average, 0.96 ± 0.8 and 0.22 ± 0.02 mg g⁻¹ FW, respectively), while no significant effect was observed in the leaf pigment concentrations of “Red Rubin” (1.13 ± 0.10 and 0.23 ± 0.02 mg g⁻¹ FW, for total chlorophylls and carotenoids, respectively; Figures 6A, B). Moreover, the A reduction observed in “Tigullio” with the 200 µM KI treatment was associated to a significantly lower concentration of carotenoids and total chlorophylls (Figures 6A, B).

Both 100 and 200 µM KI treatments determined an increase in total phenols and antioxidant capacity on mature leaves (2^nd node, Figures 6C, D). In both cultivars, the concentration of total phenols increased with the KI concentration in the nutrient solution and was not influenced by KIO₃. In the control, both the concentration of total phenols and the antioxidant capacity (DPPH assay) were much higher in “Red Rubin” than in “Tigullio” (Figures 6C, D): 6.56 ± 0.48 against 2.49 ± 0.04 mg g⁻¹ FW of gallic acid equivalents, and 0.74 ± 0.09 and 2.33 ± 0.27 mg FW ml⁻¹, respectively for the inhibitory concentration (IC₅₀). In addition to the DPPH assay, we performed also the FRAP assay on our samples: the data of the two methods showed a very good correlation for both “Tigullio” and “Red Rubin” (Pearson’s coefficients 0.994 and 0.892, respectively).

The cumulated water uptake during Experiment 4 was also measured (Figure 7A). In the control, water uptake was similar in the two cultivars when expressed per plant (Figure 7A), while it was significantly higher in “Red Rubin” when expressed on a leaf area basis (Figure 7B). For both cultivars, no differences were found among the control and 100 and 200 µM KIO₃. Compared to the control, whole plant water uptake was significantly decreased at 100 and 200 µM KI, while an opposite result was found when water uptake was expressed on a leaf area basis (Figure 7B).

In this work, KI and KIO₃ concentrations not higher than 10 µM or 100 µM, respectively, did not influence the total biomass in “Tigullio” or “Red Rubin” cultivars (Table 2; Tables SM1 and SM2). Nevertheless, the use of nutrient solution with 50 µM KI or 400 µM KIO₃ resulted in a similar reduction of plant growth (Table SM2), thus suggesting that in sweet basil I⁻ added to the nutrient solution is approximately eight-times more phytotoxic than IO₃⁻.

In both cultivars, higher concentrations of KI (100 or 200 µM) in the nutrient solution caused a significant decrease of plant height, total dry weight and leaf area compared to the control, and growth inhibition was more severe in “Tigullio” than in “Red Rubin” (Table 2; Table SM2), suggesting a higher iodine tolerance of the red-leaved cultivar. This conclusion was corroborated by the result of the application of the Maas-Hoffman model to assess the plant response to iodine supply, as a lower slope and a higher critical concentration (the threshold) was computed for “Red Rubin” than for “Tigullio” (Table 3).

Several authors found a positive effect of iodine applied to the nutrient solution on plant growth, especially if this element was supplied at low concentration and/or as IO₃⁻ (Borst Pauwels, 1961 on barley and tomato; Zhu et al., 2003 on spinach; Li et al., 2017 on strawberry). For example, Li et al. (2017) reported an increase of plant biomass in strawberry when iodine was applied to the nutrient solution up to 1.97 µM as I⁻ and 2.86 µM as IO₃⁻. In contrast, detrimental effects were observed when the I⁻ or IO₃⁻ concentrations in the nutrient solution were higher than 10–40 µM or 100–200 µM, respectively (Blasco et al., 2008). In our experiments, the addition of KI or KIO₃ to the nutrient solution never produced a significant increase of total biomass with respect to the control, probably due to the much higher concentrations (10 µM or higher) tested in comparison to those reported in the literature.

A decrease in the biomass of iodine treated plants was also reported in lettuce (Blasco et al., 2012; Smoleń et al., 2014a), tomato and potato (Caffagni et al., 2011), as well as in carrot (Smoleń et al., 2014b) and in Opuntia (García-Osuna et al., 2014), although in other plant species whose vegetative reserve organs are also harvested, such as onion, iodine seemed to have no effect on plant weight (Dai et al., 2009).

Toxic iodine concentrations in the nutrient solution significantly reduced the CO₂ assimilation of mature leaves in both basil cultivars, albeit to a lesser extent in “Red Rubin,” which had a lower photosynthetic capacity compared to “Tigullio” (Figure 4A). In both cultivars, leaf CO₂ assimilation was negatively correlated to leaf iodine content (Figure 5); however, the slope of the regression line between these parameters was much lower in “Red Rubin” than in “Tigullio” and this is a further evidence of the greater iodine tolerance of the purple-leaved cultivar. In “Tigullio,” the reduction of CO₂ assimilation was attributable to both stomatal and mesophilic limitations as leaf stomatal conductance decreased while the intercellular CO₂ concentrations did not change following iodine stress (Figure 4C). On the contrary, only mesophilic limitations were observed in “Red Rubin.” Application of 80 µM KI reduced leaf CO₂ assimilation in lettuce and this effect was associated with a reduction of stomatal conductance and total chlorophyll (Blasco et al., 2011).

In “Tigullio” the detrimental effects of 100 and 200 µM KI on leaf CO₂ assimilation were associated to a reduced content of total chlorophylls and carotenoids (Figures 6A, B), which was also revealed by the occurrence of leaf chlorosis. No significant effects of iodine level were found on the leaf pigment concentrations of “Red Rubin.”

The reduction of the content of photosynthetic pigments due to toxic concentration of KI was found in barley (Duborská et al., 2018) and in lettuce (Lawson et al, 2015; Medrano-Macías et al., 2016).

The reduction of stomatal conductance observed in mature leaves of “Tigullio” following KI application was not associated with reduced water uptake as expressed on a leaf area basis, which instead increased in both cultivars (Figure 7). The cumulated water uptake of whole plants, which was accounted for 94–96% by transpiration in previous experiments with the same sweet basil cultivars (Pardossi et al., 2015), was decreased by KI application as a result of reduced leaf area.

In all the treatments, “Red Rubin” showed a higher water uptake per leaf area rate than “Tigullio” (Figure 7B), probably due to the purple colour of leaves that resulted in a higher average leaf temperature (0.6–1.0°C more than “Tigullio,” data not shown), and thus favoured the transpiration process.

Accordingly, the experiments of Voogt et al. (2010; 2014) on the iodine biofortification of lettuce, cucumber, pepper, round and cherry tomato highlighted that the application of iodine did not alter the water uptake capacity of the crop, and possibly the cumulated water uptake differences could be ascribed to the total leaf area reduction.

The growth reduction observed in both cultivars exposed to 100 and 200 µM KI was associated with a significant reduction of the leaf concentration of N, P, K (not in “Red Rubin”), and Zn (Table 4). Leaf contents of Mg, Mn, and Cu were not affected, while the contents of Fe and Ca were significantly increased. The application of KI also increased the leaf concentration of nitrate.

Similar results were found in lettuce grown in soilless culture by Blasco et al. (2012). These authors found that, compared to the control, the application of 40 µM KI decreased the leaf content of N, P, and K and increased Fe content without significant effects on the leaf concentration of Cu and Mn.

Low iodine fertilizer concentrations (100 µM KI or KIO₃) added in the irrigation water were found to increase P, Mg, Fe, K, Cu, and Mn levels in Opuntia Ficus-indica (García-Osuna et al., 2014).

In contrast with our findings, Smoleń et al. (2015a) found a negative correlation in field-grown lettuce between the concentration of iodine and those of Mg, Ca, S, Na, B, Cu, Fe, Mn, Zn, and K (the latter in agreement with our results).

Blasco et al. (2012) suggested that high concentrations of iodine may alter the specific root transporters for NO₃⁻, affect P and K uptake or trigger an antagonistic interaction that would explain the reduction of these elements in the leaves of basil plants. Schlorke et al. (2016) suggested that a high iodine content in plant tissues could induce detoxifying mechanisms that increase the activity of oxidizing enzymes and lead to changes in tissue concentrations of Cu, Fe, and Mn. Alternatively, Kato et al. (2013) suggested that IO₃⁻ may induce reductase activity in the root, which could impact on the bioavailability of mineral nutrients, and that the activation of iodate reductase could induce other responses associated with redox signalling to counteract the effect of IO₃⁻.

Indeed, in our work the leaf concentration of all considered nutrients remained within the sufficiency ranges reported for sweet basil (Zheljazkov and Warman, 2003); therefore, the remarkable growth reduction observed in plants treated with KI cannot be explained by iodine-induced mineral deficiencies. Similar conclusions were reported for barley by Duborská et al. (2018) and for lettuce by Blasco et al. (2012) and Smoleń et al. (2014a).

Sweet basil is a vegetable that can accumulate high content of nitrates (Muráriková and Neugebauerová, 2018). The application of KI also increased the leaf concentration of NO₃⁻ (Table 4). An increase in leaf NO₃⁻ content due to iodine exposure was observed by several authors (Wong and Hung, 2001; Blasco et al., 2012; Smoleń and Sady, 2012; Smoleń et al., 2014a).

Medrano-Macías et al. (2016) suggested that IO₃⁻ could act as a substrate for widespread enzymes, such as nitrate reductase. Accordingly, Smoleń et al. (2011) reported that nitrate reductase could catalyse the reduction of IO₃⁻ to I⁻, thus interfering with NO₃⁻ metabolism in plants.

Plants can absorb iodine by the roots as I⁻ through ionic channels and chloride transporters that are energized by proton pumps (White and Broadley, 2009). The lower toxicity of IO₃⁻ observed in our work and in many other species (e.g.: barley, Duborská et al., 2018; cabbage, Weng et al., 2008; lettuce, Blasco et al., 2008; Blasco et al., 2010; Voogt et al., 2010; rice, Mackowiak and Grossl, 1999; spinach, Zhu et al., 2003; Smoleń et al., 2012; tomato, Caffagni et al., 2011; Kiferle et al., 2013) could be related to the necessity for IO₃⁻ to undergo electrochemical or enzymatic reduction to I⁻ prior to plant uptake (Zhu et al., 2003; Mackowiak et al., 2005; Kato et al., 2013).

After root absorption, iodine is transported to the shoot mainly through the xylematic flux, while its redistribution through the phloem is low (Mackowiak and Grossl, 1999; Smolen et al., 2014a). Voogt et al. (2014) concluded that i) iodine accumulation in the shoot is dependent on the mass flow of water caused by plant transpiration and ii) the differences in iodine distribution across plant species and seasons could be explained by the difference in the transpiration rate.

In our work, iodine uptake and translocation to the leaves of both basil cultivars were dependent on the iodine form and concentration in the nutrient solution (Figure 3). The addition of KI to the nutrient solution produced an accumulation of iodine in the leaf tissue approximately eight-times higher than that induced by KIO₃. At the same KI or KIO₃ concentration in the nutrient solution, iodine tissue content in “Red Rubin” was similar or significantly higher than in “Tigullio” (Table 2 and Figures 3 and 4D). This is an evidence that iodine tolerance in “Red Rubin” could not be ascribed to a root iodine-excluding mechanism or to a slower xylem loading of iodine than in “Tigullio,” and suggests that the higher iodine tolerance of “Red Rubin” was probably due to a superior ability to tolerate higher concentrations of iodine in leaf tissue.

Zhu et al. (2003) in hydroponically-grown spinach treated with 100 µM KI or KIO₃, calculated the solution-to-leaf-transfer factor (TF_LEAF) as the ratio between the iodine concentrations in leaves expressed on a fresh weight basis and in the nutrient solution: it was 19.4 and 2.2 for KI and KIO₃, respectively. In our work (Experiment 3) TF_LEAF was 14.3 for 200 µM KI and 2.1 for 200 µM KIO₃, in good agreement with the values reported by Zhu et al. (2003) for spinach.

Weng et al. (2008) demonstrated by electron microscopy that in cabbage roots iodine accumulated mainly in the cell wall, while in the leaves iodine was stored in the chloroplast. Some bibliographic evidences confirmed that iodide could easily be oxidized to elemental iodine, which can disrupt the cell membranes of roots and oxidize chlorophylls and carotenoids, thus resulting in leaf chlorosis and reduced CO₂ assimilation (Lawson et al., 2015; Medrano-Macías et al., 2016; Duborská et al., 2018). Blasco et al. (2008; 2010) found that iodine application led to an increase in the concentrations of antioxidant molecules (e.g. ascorbic acid, glutathione, and phenolic compounds) and in the activity of some enzymatic antioxidants (e.g. superoxide dismutase, catalase, L-galactono dehydrogenase enzymes). In our work, we measured the concentration of total phenols and the antioxidant capacity in mature leaves, which were both higher in “Red Rubin” than in “Tigullio” and increased with leaf iodine content in both genotypes (Figures 4D and 6C, D).

Phenols and anthocyanins are secondary metabolites involved in the plant protection against different types of biotic and abiotic stress, including mineral toxicity (Pardossi et al., 2015; Medrano-Macías et al., 2016). High leaf phenolic concentrations were thought to play an important role in boron tolerance of “Red Rubin” plants (Landi et al., 2014; Pardossi et al., 2015), and phenolic compounds can bind iodine through electrophilic substitution of H in the aromatic ring (Reiller et al., 2006). The anthocyanins contained in the epidermis of “Red Rubin” could act as a photoprotective layer for leaf mesophyll that partly absorbs the incident light energy and may diminish photooxidative damages of the chloroplasts (Landi et al., 2014; Pardossi et al., 2015). Based on these evidences, we can hypothesize that the same mechanisms could be involved also in the iodine tolerance of “Red Rubin.” Further work is necessary to verify this assumption.

In Experiment 1, we found that 10 µM KI did not affect plant growth and increased the leaf iodine content to 295 and 420 mg g⁻¹ DW, respectively, in “Tigullio” and “Red Rubin” (Table 2). Considering a leaf dry matter content of 7.5% for basil leaves (on average), we calculated that the daily iodine RDA for healthy adults (150 µg day⁻¹) could be satisfied by the assumption of approximately 6.7 g of fresh biofortified leaves of “Tigullio,” which corresponds to 7–8 leaves, an amount easy to add to a portion of salad.

Green-leaved sweet basil is also widely used to prepare the worldwide popular sauce “Genovese pesto.” Using the above-mentioned data, we calculated the hazardous amount, which is the maximum daily amount of sweet basil below the recommended dietary tolerable upper intake of iodine (1,100 µg day⁻¹ for an adult of 70 kg body weight; Smoleń et al., 2019). For “Tigullio” containing 295 mg I kg⁻¹ DW, the hazardous amount is 49.7 g of fresh leaves, which is approximately three-fold higher than the amount necessary to prepare a portion of pasta (80 g) dressed with “Genovese pesto” (about 12 g of sauce containing 6 g of fresh basil leaves).