*4.6. Iron*

In human health, the main function of iron (Fe) is related to the synthesis of hemoglobin and myoglobin besides being essential to many metabolic processes such as oxygen transport, deoxyribonucleic acid (DNA) synthesis, and electron transport, it is also required for energy production [109]. The RDA of Fe ranges between 8 and 18 mg day−1, whereas the UL for adults is 45 mg day−<sup>1</sup> [69]. Iron is a versatile, essential element in plant metabolism, whose biological functions are primarily based on the reversible redox reaction of Fe2+ (ferrous) and Fe3+ (ferric) ions, the ability to form octahedral complexes with various ligands and to change its redox potential in response to different environmental conditions. Due to this, Fe is involved in the transfer reactions at the base of life, since electron transfer chains of photosynthesis and respiration rely on iron–sulfur (S) clusters of the 2Fe–2S or 4Fe–4S type [110]. The concentration of this element in soil often exceeds plant requirements, being present at 20–40 mg kg−<sup>1</sup> [111], but usually only a small amount of this is available for plant nutrition. Particularly in alkaline and calcareous soils, once applied through fertilization, Fe quickly becomes unavailable to roots absorption, because of precipitation, adsorption, and oxidation phenomena [112,113]. Plants have evolved two different strategies to acquire Fe from the growth substrate, based either on its reduction (Strategy I plants) or chelation with organic ligands (Strategy II plants) [114]. In nongraminaceous species (Strategy I plants), such as most of vegetable crops, organic acids and phenolic compounds released by roots chelate ferric Fe on the root surface (Fe3+), which is subsequently reduced to its ferrous form (Fe2+) to transport the element across the plasmalemma of root epidermal cells [27]. The Fe transportation within the plant occurs in chelated forms, mainly with citrate and malate in the xylem, and nicotianamine and its derivatives in the phloem [115]. This condition derives from the peculiarities of this metal, characterized by low solubility and high reactivity, so its transport inside the plant must be associated to proper chelating molecules controlling its redox states between ferrous and ferric forms [116]. The status of Fe into a plant is expressed by its quantity, redox state, speciation with chelating molecules, and its compartmentalization [117]. Chloroplasts represent the main pool of Fe within the cell, as they gather approximately 80–90% of cellular Fe [44]. This flows from the high Fe demand of the photosynthetic apparatus, and Fe-deficiency hampers the electron transfer between PSI and PSII, resulting in photooxidative damages [116]. Even though the range of Fe in leaves is between 50 and 150 mg kg−<sup>1</sup> DW, Fe requirement is highly variable among species. For example, C4 species are more likely to require higher Fe amounts than C3 species; fast growing meristematic and expanding tissues need more Fe. On the other hand, Fe toxicity is reported in concentrations above 500 mg kg−<sup>1</sup> DW, which can cause damages associated with formation of ROS, inducing the activity of antioxidative enzymes such as ascorbate peroxidase, besides damages to membrane and irreversible impairment of cellular structure, DNA, and proteins [44]. To improve Fe uptake agronomical solutions to make Fe available are acidification of soil [112] and/or use as Fe(III)-chelates synthetic fertilizers. Since the latter are expensive, their use is mainly restricted to soilless crops and to high added-value cash crops [117]. However, in the case of vegetable crops, the knowledge concerning Fe enrichment, and specifically biofortification, is still poor. One alternative to provide Fe to plants is the foliar spray even if, both adopting the chelated or the sulfate-salt form, a large fixation by cuticle can be observed [118]. Foliar spray of Fe sulphate heptahydrate (FeSO4·7H2O) proved to be effective to increase Fe content both in leaves and sink organs of herbaceous crops [119,120]. In tomato, leaf spray with a 9 mM FeSO4 solution increased by 3.8 times the Fe content in roots, mediated via phloem transport [121]. In a study conducted on potato, Kromann et al. [122] did not observe a positive relationship between Fe foliar spray with EDTA-chelated Fe and its concentration

in tubers, thus the authors hypothesized that the limited effect was related to the Fe form used. As shown in Table 1, biofortification of vegetables with Fe through fertilization has been tested in few species. The use of EDDHA-chelated Fe up to 2.0 mM (112 mg <sup>L</sup>−1) proved to be effective in soilless cultivation of lettuce in increasing the Fe content of the leaves from 2.31 mg kg−<sup>1</sup> FW (control) to 4.30 mg kg−<sup>1</sup> FW [123]. In addition, it has been reported that low doses of Fe can enhance the accumulation of secondary metabolites such as chlorogenic acid, β-carotene, violaxanthin, or neoxanthin, thus leading to improved functional profiles of vegetables [123]. However, the authors observed a yield reduction of about 25%, which increased proportionally with the amount of Fe added to the nutrient solution. Overall, Fe biofortification has not been investigated enough to draw a clear picture. Using sulphate or chelate forms only in some cases enhanced mineral content in the edible part of vegetables, however, the increase was coupled with a yield reduction. Concluding significant insolubilization in the soil, limited translocation into the plant and accumulation into edible organs and negative effects on yield are the main constraints in Fe biofortification.
