*4.4. Zinc*

In human health, zinc (Zn) is essential for maintaining the structure and activity of many enzymes, besides playing a key role in the synthesis of nucleic acids and proteins. It acts in cell differentiation, glucose use, and insulin secretion [81]. The RDA of Zn ranges between 9 and 14 mg day−1, whereas the UL for adults is 40 mg day−<sup>1</sup> [69]. Zinc is essential in plant metabolism, as it plays a key role in chloroplast development and function through the Zn-dependent activity of SPP peptidase and repair of photosystem (PS I) I, besides participating in enzyme activation process such as RNA polymerases and superoxide dismutase, protein synthesis and metabolism of carbohydrate, lipid, and nucleic acid [82]. Although most of the world's cultivated soils contain enough Zn to sustain its accumulation in plants' edible portions (between 10 and 100 mg kg−1), Zn phytoavailability is a factor often limiting its uptake by roots, so that it has been estimated that about one-fifth of the world's population actually suffers from Zn deficiency [83]. Under these conditions, agronomic strategies are aimed to improve the Zn phytoavailability into the soil, e.g., by correcting soil alkalinity, implementing more proper crop rotations, introducing beneficial soil microorganisms, or delivering phytoavailable Zn through the application of Zn-fertilizers to soil or foliage [83]. Zinc is absorbed by the plants from the soil solution primarily as Zn2+ (Strategy I plants) or complexed with organic ligands released by roots (phytometallophores), a mechanism which is restricted to cereals (Strategy II plants) [84]. Once inside the plant, xylem loading occurs either via symplast and apoplast, whereas in the xylem sap Zn is transported in its ionic form or in form of metal complexes with asparagine, histidine, organic acids, and nicotianamine [85]. Similarly, phloem Zn redistribution to various organs is thought to be affected either as divalent cation or in complexed forms with nicotianamine, malate, or histidine [27]. Due to its low phloematic mobility, Zn-supplied plants through the rhizosphere show a decreasing Zn concentration in the order shoot ≈ root > fruit, seed, tuber, thus showing a penalty on phloem-fed organs [86]. For this reason, root crops and leafy vegetables are thought to have the greater potential to increase dietary Zn uptake [83]. It must be pointed out that despite the low Zn phloematic mobility, Zn translocation through phloem for several plant species after application to foliage has been found to be nutritionally considerable for their growth and development, especially when cultivation occurs on substrates with low Zn phytoavailability [87]. Plants markedly differ in their ability to accumulate Zn in their tissues, but as a general rule, most crops require a leaf Zn concentration higher than 0.015–0.030 g kg−<sup>1</sup> DW to reach their maximal yield. However, phytotoxicity symptoms are usually noticed at concentrations greater than 0.1–0.7 g kg−<sup>1</sup> DM, depending on the species and exposure time [83]. When toxicity levels are attained, plants show an array of heavy metal stress responses such as growth and yield inhibition, leaf chlorosis and necrosis, restricted stomatal conductance and CO2 fixation, changes in chlorophyll structure and concentration [88], so the higher threshold concentration actually represents a physiological limit to the biofortification achievements. Nonetheless Zn hyperaccumulation capacity has been observed in members of Brassicaceae, Caryophyllaceae, Polygonaceae, and Dichapetalaceae, whereas a greater Zn susceptibility has been noticed in the Linaceae, Poaceae, and Solanaceae [84]. Common inorganic Zn-fertilizers include ZnSO4, ZnO, and synthetic chelates [27] such as Zn-EDTA, Zn-DTPA, or Zn-HEEDTA. When foliar applications are concerned, the Zn compounds used must be highly soluble and enter rapidly into the leaf apoplast, in order to promote Zn translocation to phloem-fed organs, so avoiding possible interferences with mesophyll metabolism [86]. Due to their ability to hyperaccumulate Zn, leafy *Brassicas* have been extensively studied in biofortification protocols (Table 1). In kale leaves (*Brassica oleracea* L. var. *acephala*), de Sousa Lima et al. [89] reported up to a 28-fold increase of Zn concentration by providing the crop with 300 mg Zn kg−<sup>1</sup> soil. After applying 22.7 kg ha−<sup>1</sup> of Zn (in the form of Zn sulphate, ZnSO4·7H2O) to the soil, Mao et al. [90] observed a significant increase in the Zn concentration of the edible portions of canola (*Brassica napus* L.) and cabbage (*Brassica rapa* L. Chinensis Group) (by 25% and 200%, respectively). Zinc biofortification through foliar spray has been successfully performed in arugula (*Eruca sativa* L.) using 1.5 kg ha−<sup>1</sup> of ZnSO4·7H2O, with a resulting +94% increase of leaf Zn concentration [91]. Among non-Brassicas leafy vegetables, in a study conducted by Barrameda-Medina et al. [92] hydroponically cultured plants of lettuce (*Lactuca sativa* L.) supplemented with 100 μM ZnSO4·7H2O in the nutrient solution showed a 251% increase in leaf Zn concentration. Simultaneously biofortification programs must take into account that high Zn concentration

on soil cultivated crops can negatively affect Fe absorption and improve the content of Mn and of amino acids [89]. In conclusion, Zn biofortification, especially in the form of sulphate is promising in increasing the mineral content in vegetable products.
