*4.1. Calcium*

In human health, calcium (Ca) is required in several systems, like musculoskeletal, nervous and cardiac. It is essential to maintain good bones, teeth, and mineral homeostasis. It also acts as a cofactor in many enzyme reactions and contributes to the function of the parathyroid gland [42]. The RDA of Ca ranges between 1000 and 1300 mg day−1. The UL for adults is 2500 mg day−<sup>1</sup> [43]. Calcium is an important nutrient for plant metabolism, involved in structural functions of cell, acting as a counter-cation for organic and inorganic anions trafficking across the tonoplast and as an intracellular, cytosolic messenger [44]. It is one of most abundant nutrients in the earth's crust, with an average concentration of about 36.4 g kg−1[45]. Ca2+ concentration in the soil solution is usually enough (0.1–20 mM) to meet the plants' demands or, in neutral and calcareous soils, to exceed their requirement, thus leading to Ca accumulation in the vicinity or inside the roots [44]. However, some Cadeficient conditions can sometimes be encountered, especially in highly weathered tropical soils or in saline/sodic soils. Calcium is absorbed as divalent cation by the root apex and/or regions of lateral shoot initiation [46], where Casparian band between endodermal cells is absent or disrupted, and/or the endodermal cells surrounding the stele are not suberized [47]. Once inside the plant, Ca moves primarily through the xylem [46] with the water flow driven by transpiration [48,49], either as Ca2+ or complexed with organic acids [50]. However, Ca2+ movement inside the xylem vessels cannot be explained simply in terms of mass flow, as Ca2+ ions are also absorbed by adjacent cells and are complexed to nondiffusible anions in the xylem walls [48]. Due to its slow phloematic mobility, this element is present at lower concentrations in mostly phloem-fed organs (e.g., young leaves, fruits, and tubers) than in the older leaves ( ≈10-times less). Considering the mineral partitioning inside the plant, leafy vegetables can play a primary role in the dietary intake of Ca, so being possible targets for Ca biofortification [51]. This last point should be addressed at increasing the Ca content of the edible portions, without adversely impacting both plant growth and production costs [27]. Most plant species can accumulate high Ca contents in leaf laminae (up to 100 g kg−<sup>1</sup> DW) without any symptoms of toxicity, because Ca exceeding plant's needs is detoxified by sequestering as insoluble Ca oxalate and deposited either in the cell wall or stored inside the vacuole [44,47]. Depending on the plant species, tissues, and growing conditions, Ca concentration in plants varies between 1 and >50 mg kg−1. However, some species may have insufficient detoxification mechanisms, so their growth can be severely depressed at high Ca tissue content [44]. Excessive Ca can cause toxicity symptoms such as the presence of yellow flecks formed by crystals of calcium oxalate and growth inhibition, the latter can be observed even in calcicole species (plants occurring in calcareous soils) when submitted to a soil solution

with a concentration higher than 10 mM Ca [46]. Strategies for Ca biofortification should include (i) increasing Ca supply to cells; (ii) increasing Ca uptake into cells; (iii) removing compounds making Ca unavailable and/or (iv) increasing Ca storage at the cellular and/or tissue level [27,52,53]. The application of Ca fertilizers can increase its concentration mostly in leafy vegetables (Table 1), whereas for grain, seeds, and fruits, sound indications are still to be reached. In 21-day old *Brassica rapa* plants grown on soil, the increased Ca supply to roots (compost mix supplemented with 0.4 vs. 3.5 g CaCl2 <sup>L</sup>−1) significantly enhanced its concentration in leaves (0.75 and 25 g kg−<sup>1</sup> DW, respectively). The result was not influenced by the different supply of Mg fertilizer [54]. To reduce the effects of different soil characteristics (e.g., minerals concentration, pH) on Ca availability, soilless cultivation on inert substrates or water (e.g., floating system) allows a better control of the ion concentration in the root environment. In some leafy vegetables, D'Imperio et al. [24] increased the Ca concentration by adding calcium phosphate and calcium chloride in the nutrient solution (from 100 to 200 mg <sup>L</sup>−1), determining an increase of Ca concentration in leaves of basil ( ≈15%) and mizuna ( ≈12%), but not in tatsoi or endive (Table 2). Moreover, the biofortification process did not influence their oxalate content nor Ca bioaccessibility. A higher Ca content (up to 5-fold higher than control) in lettuce (*Lactuca sativa* L.) grown in a floating system was obtained by Borghesi et al. [55] with a nutrient solution containing 800 mg Ca L−<sup>1</sup> (as CaCl2), compared to the control with no Ca addition. However, the high salt content increased both the Cl concentration and electrical conductivity of the nutrient solution, so reducing the marketable quality and yield ( −32%). Foliar applications of soluble Ca fertilizers are commonly made for several horticultural crops, to prevent Cadeficiency disorders. However, only few experiments refer to Ca biofortification through foliar applications. Moreover, these applications are expected to have limited effects on Ca content of roots, tubers, and seeds, because of the typical translocation patterns of the element. In one of few experiments, Yuan et al. [56] observed a significant increase of Ca concentration in lettuce sprayed three times every 20 days with 120 mg L−<sup>1</sup> of CaCl2 compared to 60 and 180 mg L−<sup>1</sup> (21.4% and 5.2%, respectively), although this effect was genotype-dependent. Overall, Ca biofortification of vegetables using Ca chloride proved to be effective in the majority of the studied leafy crops even if negative effects on yield cannot be excluded; besides, one of the main challenges is related to the presence of oxalate, which can partially limit Ca bioavailability.


**Table 1.** Response of some vegetable crops to biofortification (1).


**Table 1.** *Cont*.

(1) The list reports the most representative horticultural crops. In this and in the following tables, data refer to research on Scopus**®** using "biofortification" and "vegetables" as keywords performed in November 2020. Papers which tested more than one species were counted more than one time. (2) Calculated in the edible portion.


*Foods* **2021**, *10*,

**Table 2.** Chemical forms of each mineral used in the biofortification of some vegetable crops.

 223

initial and final concentration of mineral element in the edible part of vegetables.
