S is a macronutrient essential for plant growth and development. It is required for the biosynthesis of cysteine (Cys), methionine (Met), and glutathione (GSH) and for several secondary metabolites, such as glucosinolates, as well as for the biosynthesis of proteins, cofactors, and vitamins. S-containing metabolites have central roles in the responses of plants to various environmental conditions ^[1][2][3][18,19,20].

S is taken up from the rhizosphere in the form of sulfate, via sulfate transporters localized in the cytoplasmic membranes of rhizodermal and outer cortex cells. Subsequently, sulfate will either be transferred to the root stele and loaded in the xylem to be transported to the shoot or it will be translocated into root plastids. Upon arrival of the sulfate to the aerial plant parts, it will be finally transferred into chloroplasts. At any time, depending on the needs of the plant, sulfate may be also transported into the vacuoles of the cells either of the roots or the shoot of the plant. All these transfers are made mainly through specific sulfate transporters, localized either in a cytoplasmic membrane or in a subcellular organelle’s membrane ^[2][4][5][19,21,22].

After entering in a root or shoot plastid, sulfate is assimilated into adenosine-5′-phosphosulfate (APS), which is then reduced into sulfite, and then sulfide, leading to cysteine biosynthesis. Cysteine is the key metabolite for subsequent biosynthesis of the S-containing organic compounds in plants, while a major pool of sulfur is GSH, a Cys-containing tripeptide. In parallel, APS will be phosphorylated to 3′-phosphoadenosine-5′-phosphosulfate, an intermediate metabolite used for sulfation reactions. Moreover, sulfite may also be used for sulfolipids biosynthesis into the plastid, or it may be transported to peroxisome, where it can be reoxidized to sulfate through the activity of sulfite oxidase ^[1][2][6][7][18,19,23,24].

The regulation of S homeostasis takes place predominantly during sulfate uptake and APS reduction, through transcriptional regulation of the sulfate transporters as well as of APS reductase (APR) isoforms. Several metabolites have been appointed to have roles in the control of sulfate assimilation: GSH serves as a negative regulator through feedback inhibition, while OAS is a positive regulator of the sulfate uptake and assimilation pathway ^[1][5][8][18,22,25]. Phytohormones also play outstanding roles in the regulation of S homeostasis, such as cytokinins as well as the “stress-related” hormones abscisic acid, jasmonic acid, and salicylic acid ^[1][18]. Furthermore, the roles of regulatory components of sulfate uptake and assimilation such as the transcription factor SLIM1, the sulfur-responsive element SURE, and the miR395 have been described ^[2][19]. Recently, an epigenetic regulatory mechanism involving the nuclear-localized MORE SULFUR ACCUMULATION1 (MSA1) methyltransferase has been identified ^[9][26].

EMs play central roles in the metabolism, growth and production, maintenance, abiotic and biotic stress tolerance, and as structural and functional components of metalloproteins if they are at an optimal concentration. EMs are transition metals, with Fe and Cu to undergo redox changes under biological conditions. At supraoptimal levels, they are toxic because they can cause oxidative stress due to the production of reactive oxygen species via Fenton reaction. To sustain the appropriate ion homeostasis, plants maintain equilibrium in EM homeostasis by the establishment and maintenance of stable linkages with appropriate organic ligands in a specific geometry ^{[10][11][12][13][14]}[27,28,29,30,31]. The following structural and functional roles highlight the biological importance of each one of the EMs.

Importance of Fe—Fe is required in several cellular processes, including photosynthesis, respiration, and sulfur assimilation. Fe exists as Fe(III) or Fe(II) under physiological conditions, participating in electron transfer reactions of the cells. The bulk of Fe in plant cells is needed in mitochondria and chloroplasts, where the major sinks of Fe are Fe-S clusters and heme are, and it is stored in ferritin or in vacuoles.

Several proteins contain Fe as a cofactor, mainly in the form of Fe-S clusters. Such proteins belong to the electron transport chains of chloroplasts and mitochondria. In the chloroplasts, the major Fe-S proteins are photosystem I (PSI), cytochrome b6f complex, ferredoxin, nitrite reductase, sulfite reductase, and adenosine 5′-phosphosulfate reductase (APR). In mitochondria, the main Fe-S proteins are the complexes I, II, and III of the respiratory chain and aconitase in the citric acid cycle. The biosynthesis of these clusters requires tightly regulated provision of chelated Fe and reduced S, in the form of cysteine ^{[14][15][16][17][18]}[31,32,33,34,35].

Importance of Cu—Cu is a redox-active transition metal and under physiological conditions in vivo, it exists as Cu(II) or Cu(I) and participates in many physiological processes. Cu ions act as cofactors in a variety of enzymes such as cytochrome c oxidase, Cu/Zn-superoxide dismutase (Cu/Zn SOD), ascorbate oxidase, plastocyanin, laccase, amino oxidase, and polyphenol oxidase. At the cellular level, Cu possesses key roles in photosynthetic and respiratory electron transport chains, C and N metabolisms, biogenesis of molybdenum cofactor, Fe mobilization, protection against oxidative stress, oxidative phosphorylation, transcription protein trafficking machinery, ethylene sensing, and cell wall metabolism ^[12][19][20][29,36,37].

Importance of Zn—Zn forms tetrahedral complexes with cysteine residues of polypeptide chains. Known roles include its presence in enzymes involved in protein synthesis and energy production. Zn is required for the maintenance and the structural integrity of membranes via its binding to membrane phospholipid and sulfhydryl groups, resulting in the protection of membrane lipids and proteins against oxidative damage. It is involved in signal transduction pathways via mitogen-activated protein kinases, and it plays an important role in seed development ^[15][21][22][32,38,39].

Importance of Mn—Mn presents the oxidation states II, III, or IV, and serves as a cofactor in various enzymes within a plant cell, where it can fulfill two roles in proteins: (1) as a catalytically active metal or (2) as an enzyme activator. Mn contributes to photosynthesis, defense against oxidative stress, lipid biosynthesis, nitrogen metabolism, gibberellic acid biosynthesis, and RNA polymerase activation. Representatives for the catalytic role are the Mn-containing water splitting system of photosystem II, the Mn-containing superoxide dismutase, and the oxalate oxidase. The group of the Mn-activated enzymes consists of PEP carboxykinase, isocitrate dehydrogenase, phenylalanine ammonia lyase, and malic enzyme. Proteins of this group are known to be involved in the shikimic acid pathway, as well as the biosynthetic pathways of aromatic amino acids, flavonoids, lignins, and the indole acetic acid. Τhe role of Mn in the activation process is less specific and in many cases it can be replaced by magnesium ^[15][23][24][32,40,41].

The EM-S bonds—The first level of interaction between EM and S is the formation of an effective EM-S bond (Figure 1). Ligands are distinguished as weakly (or hard) vs. highly (or soft) polarizable ones. The weakly polarizable ligands include the carboxylate groups with negatively charged oxygen atoms and the carbonyl groups with polar oxygen atoms. The highly polarizable ligands include the sulfhydryl groups. Aspartate (Asp) and glutamate (Glu) participate with their carboxylate groups; asparagine (Asn) and glutamine (Gln) participate with their carbonyl groups; while Cys and Met, the S-containing amino acids, are soft ligands. Histidine (His) carries the imidazole ring that contains the borderline aromatic N.

The contribution of S in the bond—The S atom is a soft donor; hence, it prefers to bind with soft cations. In this way, it provides high redox potential to metal redox couples. The reduced form of the metal cation is softer than the oxidized one. Between two metal centers, the S-containing ligands can function as bridging ligands (M-S-M) or as monodentate ones (M=S). In proteins, S is found as thiolate or thioether. Thioethers are organic sulfides (C-S-C). Examples of sulfides are Met and biotin. Thiolates contain the thiol group (C-S-H).

The Fe-S bond—Ligands are electron donors and the metals’ electron acceptors. The softer Fe(II) catalyzes the Fenton reaction with hydrogen peroxide; hence, an efficient bonding is needed. Fe-S proteins are characterized by the presence of Fe-S clusters, which are found in a variety of metalloproteins. These clusters contain di-, tri-, and tetra-Fe centers and are sulfide-linked in variable oxidation states. Fe-S clusters contribute to the oxidation-reduction reactions of electron transport chains. There are several proteins containing Fe-S clusters that regulate gene expression. In most Fe-S proteins, the terminal ligands are thiolate sulfur centers from cysteinyl residues. The Fe centers are tetrahedral, while the sulfide groups are two- or three-coordinated ^[25][42].

The Cu-S bond—The harder Cu(II) can be co-ordinated by oxygen and nitrogen atoms of the harder amino acids (Tyr, Thr, His). The Cu(II)-N bonds are stable and often inert, while the Cu(II)-O bonds are more labile. The soft Cu(I) is stabilized by soft ligands. Cu(I) catalyzes the Fenton reaction with hydrogen peroxide. Cu(Ι) in proteins prefers the S atoms or ions of Cys and Met. Cu-thiolate and Cu-thioether bonds are found in a wide variety of enzymes with multifaceted co-ordination chemistry. S ligation to the metal centers provides special properties to Cu enzymes. S atoms from thiolates or thioethers act as donor ligands in a variety of Cu complexes ^[26][43].

The Zn-S bond—Zn is classified as borderline metal; i.e., Zn(ΙΙ) does not act as either hard or soft. Moreover, Zn does not have a strong preference for O-, N-, or S-coordination. In the various plant biological systems, Zn exists only as Zn(II), not taking part in redox reactions. In protein Zn-binding sites, the Zn(II) may be co-ordinated by the oxygen of aspartate or glutamate, the nitrogen of histidine, or the sulfur of cysteine. Among these, His is the most observed, followed by Cys, through which S plays a functional (catalytic) and a structural role in enzyme reactions. The Zn-S bond serves several roles: in metallothionein and Zn release, in thionein and Zn binding, in control of Zn transfer reactions and availability of cellular Zn in mononuclear sites, in the cellular distribution of Zn, in proteins that detect the availability of cellular Zn, in redox-active Zn proteins, in Zn thiolate cluster structures, and in Zn co-ordination dynamics ^[27][44].

The Mn-S bond—There are no Mn metalloenzymes containing an Mn-S co-ordination sphere, as Mn(II) establishes co-ordination with hard ligands. His is the important ligand for Mn(ΙΙ), while Cyst and Met are less likely to co-ordinate with Mn(ΙΙ) ^[23][40].

4. The Contribution of S to EM Agronomic Biofortification

The agronomic phytofortification activity to increase the EM content is associated with an increase in yield and quality. The sulfate salts of Fe, Cu, Zn, and Mn have been used for alleviating low contents of EM in seeds and edible parts, in comparison to EM complexed with synthetic ligands. The presowing application of S in the form of granular kieserite, in S-deficient soil, along with top dressing with magnesium sulphate heptahydrate and ammonium nitrate, was sufficient to achieve optimal grain yield with beneficial Fe, Mn, Zn, and Cu content in grain dry mass ^[28][29][202,203]. Fertilizer type along with application method influence the effectiveness of application on crop performance. The fertilizer formulation largely determines the EM phytoavailability. Toward increasing yield and nutritional quality of a crop, the fertilization of the rhizosphere with EM has been suggested as a sustainable strategy. However, foliar fertilization with EM often stimulates more nutrient uptake and efficient allocation in the edible plant parts compared with soil fertilization. Their combination is often the most effective method ^[30][9]. Foliar fertilization with FeSO₄ is applied when low-Fe content is found in the soil. This treatment improved grain Fe concentration in wheat by about 28% in China, and 21% in Iran, whereas in Canada, it remained ineffective. By increasing soil N application, shoot and grain Fe contents significantly enhanced both under field and greenhouse conditions ^{[31][32][33][34][35][36][37][38][39][40][41]}[204,205,206,207,208,209,210,211,212,213,214]. Application of Fe fertilizers alone either in inorganic (FeSO₄) or in chelated form (e.g., Fe-EDDHA, Fe- EDTA or Fe-citrate) presented a small positive impact on increasing Fe content in grain, while in combination with soil N application, it enhanced by 47%. At a given Fe treatment, soil N supply can enhance shoot Fe concentrations by up to 70%. Foliar application of urea also improved grain Fe concentration ^{[31][32][33][34][35][36][37][38][39][40][41]}[204,205,206,207,208,209,210,211,212,213,214]. Zn application as ZnSO₄ increased grain yield and Zn content ^[28][42][202,215]. When plants are enriched with Zn, N should be taken care of, especially during the grain pouring phase, as N plays an important role in Zn uptake ^{[43][44][45][46]}[216,217,218,219]. Zn and the other EMs concentrations increased after the use of appropriately high doses of N coupled with foliar fertilization with EMs ^{[35][45][47][48]}[208,218,220,221]. Foliar applications of MnSO₄.H₂O and CuSO₄.5H₂O, three times and separately at three critical stages of wheat grain development, i.e., middle boot, early milk, and dough stages, along with increased dose of N as top-dressing prior to heading, enhanced nutrient uptake of N, Cu, and Mn, improved yield, and enriched mineral content of the wheat grains ^[30][9]. Another phytofortification example is based on the use of elemental S. Fertilizer granules can be enriched with 2% elemental sulfur (FES). A durum wheat crop that received the enriched fertilizer accumulated a higher amount of Fe compared to a conventional one. The fertilization with FES at sowing mobilized iron, thus providing more iron to the crop, and fortified the S status of the crop, too. The initiation of the fast stem elongation stage constituted a turning point. Prior to its initiation, the use of FES increased the iron concentration in the main stems, followed by an increase in the organic S concentration. Thereafter, the FES-crop presented plants with higher main stems and fewer tillers. At harvesting, all plant parts of the FES-crop were heavier, containing more iron and organic sulfur, and the obtained commercial yield of the FES-crop was higher by 27.3% ^[49][222]. The need for integrated soil fertility management—It is highlighted that the application of sulfate salts of Fe, Cu, Zn, and Mn is effective, or more effective, under an integrated soil fertility management approach. The form of the nutrients and the interactions between them can have positive, neutral, or negative effects on yields and nutrient use efficiencies. Various factors determine the success of agronomic phytofortification, depending on EM phytoavailability at different stages, along with efficient uptake and handling of EM by the plant. Soils with (multiple) EM deficiencies are nonresponsive to NPK, despite the addition of NPK fertilizers. Management of rhizosphere N and P is important for increasing the effectiveness of the applied EM. Wheat fertilization with NP fertilizer enriched in Zn has been effective in increasing the yield of wheat grain. The adequate N and P status of plants presents positive effects on root development, EM transport to the shoot, and remobilization of EM from leaves to seeds. In wheat crops, proper N application increased Zn and Fe contents in the grain endosperm. In sorghum and finger millet crops fertilized with blends of mineral NPK enriched with Zn, B and S, the N, P, Zn, B, and S contents were increased significantly, along with crop productivity ^[35][50][51][208,223,224]. Depending on the amount of the phytoavailable Zn, the rhizospheric P can stimulate root growth and Zn uptake, or the same application of P fertilizer can trigger Zn deficiency by precipitating the already hectic concentrations of Zn, as Zn phosphate is insoluble. Addition of P appears to induce Zn deficiency through dilution effects and interference with Zn translocation from the roots. Symbioses with arbuscular mycorrhizal fungi (AMF) increases the uptake of nutrients, such as P, Fe, and Zn, that are sparingly soluble in rhizosphere. Mycorrhizal symbiosis modifies plant demands for reduced S and regulates the uptake, distribution, and assimilation of the sulfate accordingly ^[52][225]. Sulfate possibly regulates the expression of ZmNAS1, ZmNAS3, and ZmYS1 genes, revealing its potential role as signal molecule for the Fe homeostasis in AMF plants. The symbiosis with AMF prevented Fe-deprivation responses in the S-deprived maize plants. It seems that Fe was provided directly to the mycorrhizal plants through the fungal network ^[53][226]. Moreover, it has been proposed that the gene expression of the DMA exporter ZmTOM1 can be used as an early indicator for the establishment of a mycorrhizal relationship in maize ^[54][227]. The Fe uptake pathway seems to be regulated by sulfate supply in S-deprived maize plants. Moreover, a strong correlation seems to exist between the transcriptional regulation of the Fe-uptake pathway genes and the sulfate phytoavailability, and this holds true independently of the existence of mycorrhizal association or not. Sulfate is probably a key component of the signal transduction pathway that regulates the expression of the Fe-uptake pathway genes in maize plants ^[55][228].