*3.1. SULTR3;3*

In all analyzed species, one putative ortholog belonging to the SULTR3;3 group was found by BLAST analysis of HvST or OsSULTR3;3 against the different genomes, except for soybean, in which two different genes have been identified (Table 2, Figures S3 and S4). Indeed, this is not unexpected, as soybean underwent an ancient event of genome duplication [85]. The SULTR3;3 gene structure is quite conserved among species and consists of 13 exons in the majority of the genes, with the exceptions of barley and maize with only 12 exons. All genomic sequences are characterized by the presence of a long fourth or fifth intron, as reported in Figure 3a, where the structure of the characterized OsSULTR3;3 and HvST and of PvSULTR3;3 is shown as an example.


**Figure 3.** (**a**) Gene structure of *OsSULTR3;3, HvST* and putative *PvSULTR3;3* genes. Light and dark blue rectangles represent UTRs and coding exons, respectively, the black bars correspond to introns. Table 1. a legend. (**b**) Predicted domains of the SULTR3;3 protein. The sulfate transporter and the anti-sigma factor antagonist (STAS) domains are represented in red and green, respectively. Picture reproduced from [84]. (**c**) Distances between SULTR3;3 proteins, expressed as the percentage of identity. Phylogenies were constructed as described in Figure 1c.

Predicted domains of SULTR3;3 proteins are represented in Figure 3b and correspond to a sulfate transporter domain and an anti-sigma factor antagonist (STAS) domain, as previously reported [84]. Protein length varies from 647 aa of PvSULTR3;3 to 661 aa of OsSULTR3;3 (Figure S4).

Protein identity is generally very high among different species, ranging from 84.1% to 86.5% among the considered cereals and from 90.9% to 91.7% among legumes, and at 97.9% in the two paralogs of soybean, as shown in the Figure 3c diagram.

In the case of the OsSULTR3;3 gene detailed expression analysis was reported: Transgenic lines harboring the promoter of this gene fused to the GUS reporter gene revealed that a strong GUS activity was present in vascular bundles of shoots, leaves, flowers, and grains, where it was mainly detected in the scutellum. Moreover, the subcellular localization was defined to be in the endoplasmic reticulum [84]. Interestingly, both GmSULTR3;3a and GmSULTR3;3b are expressed in leaves and flowers, while only GmSULTR3;3a was expressed in the seed, with an increasing expression during seed development with a peak at 35 DAF (data not shown, in silico analysis performed using the soybean eFP Browser [47]).

The exact function of this family of proteins is still unknown and in the case of OsSULTR3;3, which was the only one analyzed in detail, no activity was revealed for the transport of phosphate, sulfate, inositol or inositol 1,4,5 triphosphate by heterologous expression in either yeas<sup>t</sup> or Xenopus oocytes [84].
