*4.8. Silicon*

Accumulating evidence from the last 30 years strongly suggests that silicon (Si) plays an essential role in bone formation and maintenance, improving the bone matrix quality and facilitating its mineralization. Increased intake of Si has been associated with increased bone mineral density and decreased osteoporosis [134]. Average daily dietary intake of Si is 20–50 mg for European population, the RDA has not been stablished; however, safe upper levels for humans have been recommended with a maximum range of 700– 1750 mg day−<sup>1</sup> [135]. Silicon is considered not essential for plant nutrition, but its inclusion in fertilization programs has proved to increase the crop tolerance to biotic and abiotic stressors [136], crop yield [137], or improve the absorption of macro- and microelements [138]. Silicon concentration in soil can vary depending on the type of soil. For example, alkaline soils containing sodium carbonate usually present a higher Si content. On average, the concentration of Si in soil is between 0.09 and 23.4 mg kg−<sup>1</sup> [139]. If compared with other minerals, Si metabolism is still poorly understood. It seems that two main mechanisms of Si absorption coexist in plants, i.e., active and passive, whose relative contributions depend upon both plant species and external Si concentration [140]. This would explain the strong differences in Si concentration reported within tissues of different plants species [141]. In any case, Si is taken up by the roots as monosilicic acid with the involvement of channels belonging to the aquaporins' group, so the water flow resulting from leaf transpiration seems to play a determinant role in defining the rate of Si absorption and transport within the plant [142]. Once absorbed, monosilicic acid is subsequently translocated to the shoot through the xylem flow, where Si is concentrated thanks to transpiration and polymerized to silica (SiO2), then deposited in the different tissues [143]. It has been reported in the Poaceae leaves that Si can be deposited both in mesophyll and epidermal cells, suggesting the coexistence of negative (transpiration-driven) and positive (though specific carriers) mechanisms controlling the Si accumulation process [144]. Plants markedly differ in their ability to accumulate Si in their various organs; concentrations ranging between 5 and 50 g kg−<sup>1</sup> DW have been reported as critical for some species. The species with low mobilization capacity accumulate it in the roots and stems, while the species with high mobilization capacity accumulate Si in stems, leaves, fruits, and seeds [142]. Gao et al. [145] noticed that excessive Si supply (>2 mM) caused the formation of Si polymers on root surfaces, a feature that could affect nutrients uptake. In spite of the scarcity of available information, this aspect would deserve extensive study with reference to vegetable crops, due to their potential role as Si source in the human diet. Indeed, thanks to their usually low silicification capacity, vegetable crops are expected to contain high amounts of soluble

Si, which is theoretically more available to be assimilated after ingestion, so potentially being optimal candidates as Si source in the human diet [142]. As shown in Table 1, as regards the leafy vegetables, in a study concerning six crops grown in a greenhouse floating system, namely *Brassica rapa* L. (tatsoi and mizuna group), *Ocimum basilicum* L., *Portulaca oleracea* L., *Cichorium intybus* L. and *Beta vulgaris* L. ssp. *vulgaris*, D'Imperio et al. [146] found an increased Si content in plant tissues by providing them up to 100 mg L−<sup>1</sup> Si (as potassium metasilicate) in the nutrient solution, with basil reaching the highest content of Si (293 mg kg−<sup>1</sup> FW, expressed as SiO2). Moreover, the authors found that Si became bioaccessible in all the considered species, in a range from 23% (basil) to 64% (chicory). In a different experiment concerning two leafy vegetables, namely chard (*Beta vulgaris* L. var. *cicla*) and kale (*Brassica oleracea* L. var. *acephala*) grown in a hydroponic system, De Souza et al. [147] compared the effects of two Si sources, namely potassium silicate and stabilized sodium potassium silicate with sorbitol, and four Si concentration in a foliar spray solution (from 0.00 to 2.52 g <sup>L</sup>−1). They found that in both species, the Si concentration in leaves linearly increased in response to Si concentration in the foliar spray solution, with the best biofortification results obtained by spraying potassium silicate. In a study concerning the green bean (*Phaseolus vulgaris* L.) cultivated in a hydroponic system, Montesano et al. [148] found that biofortified pods (obtained by adding 3.6 mM of Si as potassium metasilicate to a standard nutrient solution) showed a 310% increase of Si (from 853.8 to 2496.3 mg kg−<sup>1</sup> DW) when compared to unbiofortified ones. Moreover, they found that the bioaccessibility of Si in biofortified pods was higher than control pods (25.1% vs. 7.6%), even after cooking them by steaming or boiling. The Si biofortification protocol of strawberry fruits (*Fragaria* × *ananassa* Duchesne ex Rozier) was studied by Valentinuzzi et al. [149], who cultivated for 16 weeks in a hydroponic system provided with a standard nutrient solution, or with nutrient solutions enriched with 50 or 100 mg L−<sup>1</sup> of Si (as Na2SiO3). The authors found that providing 100 mg L−<sup>1</sup> of Si allowed to maximize the metalloid concentration in strawberry fruits (which increased from 6.44 up to 85 g kg−<sup>1</sup> DW) without compromising crop yield. However, the they observed a decrease in total phenols and an increase in the content of flavonols in response to the highest Si supply. Overall, biofortification with Si using K silicate proved to effectively increase the mineral content in vegetables. In addition, its possible role as plant protector and its ability to improve the mineral status of the plant, both make Si a key element in biofortification programs.

#### **5. Discussion and Future Trends**

The evidence discussed above pointed out that biofortification should be contemplated as a promising strategy to face malnutrition in many circumstances. Biofortification can help to obtain products designed according to the needs of two categories of target consumers (Figure 1). The first concerns products enriched with minerals that can fulfil specific functional needs; this is the case of vegetables richer in one or more minerals to counter the deficiencies related to ordinary diet or new consumer habits. (e.g., vegans). Besides vitamins, in fact, vegan diets feature an inadequate content of calcium, potassium, iron, iodine, and magnesium [150]. A second target concerns products with premium quality or superfood aimed at improving health as a whole. This would satisfy the need of an increasing group of health-conscious consumers who look at plant-based foods, especially vegetables, as a sort of medicine to prevent the insurgence of chronic diseases.

Agronomic biofortification is comparatively simpler than other methods and potentially suitable for immediate results. However, the available studies on agronomic fortification of vegetables are of a considerable number only for few crops (e.g., lettuce, tomato, spinach, and *Brassica* spp.) and for few mineral elements (e.g., selenium, iodine). For these elements, aspects related to the form, application modality, concentration, and timing have been clarified for most important crops. For all the considered elements, and particularly for selenium and iodine, the biofortification adopting soilless crops or on soil fertigated crops have been mostly considered. In some cases the model describing the accumulation in relation to the application has also been described [151]. For some other

mineral elements considered in this review, important as well in human nutrition (e.g., Fe), information is still lacking.

On the other hand, even when empirical evidence on biofortification showed a significant increase in the concentration of the mineral elements, the fortification is not economically worthwhile. In addition, an effective biofortification protocol is based on regular and frequent applications and a negative environmental impact cannot be excluded [32]. Besides, the step between biofortification and plant toxicity effects can be narrow and applications targeting the accumulation of essential micronutrients must be adjusted to avoid negative effects on plant growth [38].

**Figure 1.** Key aspects to be considered in the agronomic mineral biofortification.

The application of biofortifying elements poses some problems related to the interaction with other factors at soil level (e.g., phytoavailability) and at plant level (e.g., competition with other elements) [152]. In many studies the traditional fertigation approach is adopted, rather than foliar spray, which can be more cost effective and environmentally friendly. Indeed, foliar fertilization represents the simplest and fastest method for the application of mineral elements used for the biofortification of vegetables; but, the effectiveness depends on the used plant organ and the mobility of the element inside the plant. To face some of these problems, technical innovations such as precision agriculture, soilless cultivation, etc., may help in defining more efficient biofortification protocols.

There are only few biofortified vegetable products already present on the market (e.g., selenium enriched potato, carrot and onion, 'Selenella' from Consorzio Patata Italiana di Qualità Soc. Cons., IT, iodine biofortified potato, 'Iodì' from the Pizzoli group, IT, selenium enriched brussels sprouts from Marks and Spencer, UK, etc.). It is clear that mostly iodine and selenium have been commercially considered as biofortification elements, probably because a more efficient accumulation system and for their lower toxicity at plant level. In the future, besides a broad choice of diversified vegetables, it is expected that the market will have biofortified products richer in more than one mineral. Therefore, research that comprises simultaneous biofortification is essential. In addition, further elements are

being studied and are expected to be object of biofortification in the future (e.g., lithium, vanadium, etc.). In this regard, biofortification using Li-sulfate and Li-hydroxide was effective in increasing Li content in lettuce plants [153].

Based on the results in literature, biofortification is not expected to fully control mineral element deficiencies or eradicate them, but it complements other interventions to provide micronutrients to people. To be effective, a biofortification program should be based on very appropriate planning concerning: health and nutrition investigation, nutritional habits, design and validation of sustainable biofortification protocols, estimation of positive effect on health. Concerning biofortification protocols, the attention should be paid to those crops having an element content high enough to be conveniently targeted, and that prove to significantly benefit from mineral elements application.

In the reviewed literature most attention has been posed on the content of specific elements in plant edible portion but key concepts like bioaccessibility and bioavailability were seldomly considered. The first regards the nutrient fraction released from the food and available for absorption by the intestinal cells, while the latter expresses the amount of nutrients actually absorbed and therefore available for utilization in physiological functions [154,155]. While macronutrients (proteins, carbohydrates, and fats) are degraded and absorbed by specific and well-known biochemical mechanisms, phytochemicals and minerals are absorbable without biotransformation and often without a specific carrier [156,157]. The consequence of this poorly developed intestinal transport system is that the actual absorption of phytochemicals and minerals is deeply dependent on the food matrix. To modulate mineral bioavailability, attention should be devoted to those substances (e.g., vitamin C, β-carotene, oxalic acid, polyphenols, etc.) stimulating or inhibiting bioavailability [27,158]. Furthermore, some chemical bonds with other components in the food or the physical entrapping inside intact plant cell walls can dramatically decrease the bioaccessible and bioavailable fractions of phytochemicals and minerals [159].
