*4.7. Copper*

In human health, copper (Cu) importance is related primarily to enzymes function, contributing also to maintain cardiovascular integrity, lung elasticity, normal development of connective tissue and nerve coverings, neovascularization; it has also neuroendocrine and immune functions and it is involved in the Fe metabolism too [124]. The RDA of Cu ranges between 1.0 and 1.6 mg day−1, while the UL for adults is 10 mg day−<sup>1</sup> [69]. Copper is a redox-active transition metal that under physiological conditions exists as Cu2+ and Cu+ [125]. In plants, it is essential to many physiological processes like photosynthesis, respiration, C and N metabolism, and protection against oxidative stress. It acts as cofactor of numerous proteins and in plants it is mainly present in complexed forms, the concentration of free Cu2+ and Cu+ in the cytoplasm being minimal [44]. The worldwide average Cu concentration in soils is 14 mg kg−1, while in Europe the average concentration is 12 mg kg−<sup>1</sup> [126]. Copper is mobile in soils and its absorption is directly related to its concentration in the soil solution [44]. Plants can absorb Cu in huge amounts by roots and in minor amounts by shoots and leaves [127]. Mechanisms involved in Cu uptake are supposedly similar to those of Fe. Copper chelate reductases are encoded by ferric reductase oxidases 4 and 5 and Cu reduction occurs at the roots (Strategy I plants) where Cu is absorbed and transported by proteins of the COPT family. Copper uptake from soil depends almost exclusively on the protein COPT1, while COPT2 could act in the processes of Cu and Fe homeostasis and phosphate metabolism [27,128]. Plants can also absorb Cu through leaves, as observed by Stepien and Wojtkowiak [129] that after treating wheat plants with a foliar fertilization of copper sulphate in the amount of 0.2 kg Cu ha−<sup>1</sup> (1% CuSO4 solution) obtained a 13% increase in the Cu content. On the other hand, the redox-active transition characteristic of Cu that makes it essential also contributes to its toxicity, since the reduction between Cu2+ and Cu+ catalyzes the production of toxic hydroxyl radicals that can damage DNA, cell membranes, and other biomolecules. Besides, damage to cell membranes can be reflected in low uptake of ions and water, so Cu toxicity can be indirectly expressed as growth inhibition and chlorosis caused by the generalized deficiency of nutrients and water [130]. Normally, crop species can tolerate a maximum of 20–30 mg kg−<sup>1</sup> DW of Cu in leaves, but Cu-tolerant species can accumulate as much as 1000 mg kg−<sup>1</sup> DW of Cu in leaves [44]. Moreover, foliar fertilization of Cu in maize should not exceed 100 g ha−1, since at higher doses, between 200 and 600 g ha−1, Barbosa et al. [131] noticed phytotoxic effects that caused growth and yield reduction up to 19% and 75%, respectively. In agriculture, Cu has been used for plant disease control for decades, a number of Cu formulations have been used as biocides to contain pathogens such as bacteria, fungi and in some cases, even invertebrates. In high concentrations, Cu interacts with nucleic acids, disrupting cell membranes of pathogens. In addition, direct application of Cu is used for seed treatment, to prevent seedling infections [132]. As shown in Table 1, among the few experiences in the

biofortification of copper, Obrador et al. [133] conducted a study with spinach (*Spinacia oleracea* L.), var. 'Viroflay Esmeralda' applying eight different liquid fertilizers to the soil surface, with the irrigation water in a concentration ranging from 0 to 3 mg Cu kg−<sup>1</sup> soil. Total Cu concentration in the dry matter of shoots increased by up to 450%, from 9.55 mg kg−<sup>1</sup> (control treatment) to 52.51 mg kg−<sup>1</sup> in the treatment where plants were submitted to 3 mg Cu kg−<sup>1</sup> soil (as Cu-EDTA), a 4.54-fold increase (Table 1). However, at this dose they also noticed a 10% decrease in the dry matter yield. Instead, the dose 1 mg Cu kg−<sup>1</sup> soil resulted in an increase in Cu content of 153% allowing also to obtain a yield increase of 71% when compared to the control. Regarding the chemical form, their results showed that the best fertilizers to increase Cu content in the edible part of spinach are Cu-DHE (Cu-diethylenetri-aminepentaacetate-N −2-hydroxyethyl-ethylenediaminetriacetate-ethylenediamine-tetraacetate) and especially Cu-EDTA. Curiously, in this study, even though the total concentration of Cu in spinach shoots was higher than the maximum concentration usually tolerated by plants, no visual phytotoxic symptoms and significant yield reductions were observed. In conclusion, Cu biofortification proved to be effective using different chelate forms and its potential as a biocide could benefit biofortification programs. In addition, when Cu biofortification is concerned attention must be made to the release of Cu in the soil substrate in relation to crop rotations and soil biological properties.
