*3.2. SULTR3;4*

In the case of the SULTR3;4 group of transporters a similar situation to the one previously described for MRP proteins is present: In cereals, only one protein for each species can be found by BLAST analysis of OsSULTR3;4 against the different genomes, while in legumes, two or four paralogous proteins are present in common bean and soybean, respectively (Table 2 and Figures S3 and S5). The gene structure differs between cereals with 10 exons (the barley sequence present in the Phytozome database is incomplete with only eight exons) and legumes with 13 exons and also in this case, the fourth (the fifth in maize) intron is quite long. In Figure 4a the structures of the characterized OsSULTR3;4 and of putative ZmSULTR3;4 and PvSULTR3;4a genes are given as examples.

**Figure 4.** (**a**) Gene structure of *OsSULTR3;4* and putative *ZmSULTR3;4* and *PvSULTR3;4a* genes. Light and dark blue rectangles represent UTRs and coding exons, respectively, the black bars correspond to introns. The gene structure was obtained as described in Figure 1a legend. (**b**) Distances between SULTR3;4 proteins, expressed as the percentage of identity. Phylogenies were constructed as described in Figure 1c.

Predicted domains are the same as those already described for SULTR3;3, represented in Figure 3b. Protein length varies from 648 aa of the soybean protein to 670 aa of the rice one (Figure S5).

Also for SULTR3;4 proteins, identity is quite high ranging from 78.8% to 81.3% among cereals, from 83% to 91.9% among legumes and from 61.3% to 67% between cereals and legumes, as shown in the diagram in Figure 4b.

Analysis of the phylogenetic tree (Figure S3) clearly shows a separation between monocotyledons and dicotyledons. Furthermore, in the two legume species, the gene is duplicated, with soybean carrying four genes arising from an ancient event of genome duplication [85].

qRT-PCR expression analysis of OsSULTR3;4 gene revealed that during grain filling it was mainly expressed in node I, a very important hub for mineral distribution to upper node and panicle in Poaceae [86]. Moreover, immunostaining against GFP in lines harboring OsSULTR3;4 promoter fused to GFP, showed the highest staining in the xylem region of both enlarged- and diffuse-vascular bundles of the basal node and in node I, as well as in the parenchyma tissues between them, but not in the phloem region [20]. The activity of OsSULTR3;4 as an influx plasma-membrane localized H+/Pi symporter was shown in proteoliposomes as well as in Xenopus oocytes. Particularly, it was found that OsSULTR3;4 is involved in the intervascular transfer of P at the nodes, unloading P from xylem towards phloem [20].
