**2. Sulfate Transporters: A Short Overview**

S is an essential nutrient for plants. It is found in the amino acids cysteine and methionine, which are essential components of proteins and peptides, in vitamins and cofactors, and in a plethora of secondary compounds. S plays important and critical roles in a wide variety of cellular processes involved in plant development and response to environmental changes [6–9].

Sulfate (SO4 <sup>2</sup>−) ions in the rhizosphere are the major source of S for plants. They are absorbed by roots and then allocated to di fferent sinks by mean of specific sulfate transporters (SULTRs). The oxidized S atom in SO4 2− is then reduced and assimilated into cysteine, before entering other metabolic pathways, or directly used for sulfation reactions [9–11]. SULTRs are classified as H+/SO4 2− co-transporters, are integrated into membranes by 12 membrane-spanning domains, and contain a carboxyl-terminal region, named STAS (Sulfate Transporter/AntiSigma-factor), which is thought to be critical for both activity and stability of the transporters, as well as for their interaction with other proteins [9,12–14].

A multigene family encodes plant SULTRs. In the best-characterized species—*Arabidopsis thaliana* and, to a lesser extent, rice (*Oryza sativa* L.)—12 *SULTR* genes have been reported [14,15]. SULTRs can be divided into four functional groups or subfamilies, according to their amino acid sequences. The members of each group have specialized functions for SO4 2− uptake and distribution within the cells and among plant organs, as indicated by their di fferent tissue and subcellular localization, and regulation pathways.

Group 1 of the family encodes high-a ffinity SULTRs. Two members of this group, SULTR1;1 and SULTR1;2, are mainly expressed in the outermost cell layers of the root (root hairs, epidermis, and cortex), where they largely contribute in determining the rate of SO4 2− uptake. Arabidopsis *sultr1;1sultr1;2* double-knockout lines are severely impaired in growth and unable to take up SO4 2− at low external concentrations [16–19]. Although these transporters seem to share the same function, they are di fferently regulated to fulfill the plant demand for S-containing compounds under di fferent SO4 2− availabilities or soil conditions. In the currently accepted model, SULTR1;2 is thought to be the major component of the SO4 2− uptake system under normal S supply, whereas SULTR1;1 should play a most significant role under S deficiency or during other stresses [16,17,20–22].

Sulfate ions absorbed by root are translocated to shoot throughout the xylem and then distributed to di fferent sink organs and tissues. It has been proposed that SULTR2;1, a low-a ffinity SULTR expressed in pericycle and xylem parenchyma, may play a pivotal role in controlling the amount of SO4 2− available to be loaded into the xylem, by acting as a scavenger reabsorbing the excess of the anion in the apoplastic space inside the root stele. Under S starvation, the increase in the transcript level of *SULTR2;1* could help in maintaining adequate fluxes of SO4 2− directed to the xylem [16,23]. It is important to note that a local expression of *SULTR2;1* has also been observed in the xylem parenchyma and phloem cells of the leaves, and that it is not possible to rule out that *SULTR2;1* transcript is also expressed below detection levels in the phloem companion cells of the root [16,24].

An interesting regulatory circuit controls SO4 2− translocation and partitioning at the post-transcriptional level (Figure 1). The *SULTR2;1* mRNA is targeted and degraded by the miRNA-395 (miR395), which accumulates under S deficiency mainly in the companion cells of the phloem of both root and shoot [24]. The induction of miR395 is, in turn, activated by *SLIM1*/*EIL3* (*SULFUR LIMITATION 1*/*ETHYLENE-INSENSITIVE3-LIKE3*), a major regulator gene belonging to the EIL family transcription factors, which controls the expression of several S-responsive genes [25,26]. The mechanism by which miR395 controls *SULTR2;1* transcript level is not conventional, since the accumulation of both miR395 and *SULTR2;1* mRNA is induced under S starvation. However, the non-overlapping spatial expression domains of the two transcripts allows miR395 to restrict the expression of *SULTR2;1* to the xylem parenchyma cells of the root, thus inhibiting long-distance SO4 2− transport to sink tissues via the phloem and facilitating, at the same time, xylem SO4 2− translocation to the leaves [24,25].

**Figure 1.** Main regulatory circuits controlling SO4<sup>2</sup>− distribution in response to P or S status. Under S deficiency, the induction of *SULTR2;1*, in xylem parenchyma cells, and miR395, in phloem companion cells, enhances root-to-shoot SO4<sup>2</sup>− translocation. In this condition, the co-expression of *SULTR3,5* could help the activity of SULTR2;1 in reabsorbing the excess of SO4<sup>2</sup>− in the apoplastic space of the root. Under P deficiency, an extra regulatory circuit involving *PHR1* allows changes in SO4<sup>2</sup>− to support sulfolipids biosynthesis.

Another low-affinity SULTR belonging to group 2, SULTR2;2, seems to be involved in controlling the source-to-sink distribution of SO4<sup>2</sup>− inside the plant. Localization analyses indicate that SULTR2;2 may play a role in the transport of SO4<sup>2</sup>− via root phloem, as well as in the distribution of the anion from leaf vasculature to the leaf palisade and mesophyll, which are thought to be the primary sites for SO4<sup>2</sup>− assimilation [16]. Finally, long-distance transport of SO4<sup>2</sup>− from source to sink organs could also involve SULTR1;3, a high-affinity SULTR of group 1, as indicated by the peculiar expression of this transporter in sieve elements and companion cells of the phloem [27].

Inside the cells, SO4<sup>2</sup>− is further transported into the vacuole and chloroplast/plastid, where it is compartmentalized as S store or reduced and assimilated into cysteine for further metabolic processes, respectively. To date, tonoplast proteins mediating vacuolar SO4<sup>2</sup>− influx have not been identified. On the other hand, SULTR4;1 and SULTR4;2 are known to be involved in downloading SO4<sup>2</sup>− from the vacuoles under S limiting conditions [28].

Recently, all five members of group 3 have been indicated as redundantly involved in SO4<sup>2</sup>− uptake across the chloroplast envelope membrane [29,30]. However, these observations do not appear to be conclusive, since several other functions could be postulated for these transporters on the base of observations that are crucial for our dissertation about the hypothetical links between *SULTRs* and *lpa* phenotypes. It is important to note that if, on the one hand, reasonable uncertainties about the capacity of both SULTR1s and SULTR2s to selectively move SO4<sup>2</sup>− do not exist, on the other, no direct evidence has been provided about the actual SO4<sup>2</sup>− transport activity of most of the SULTR3 subfamily members [14]. A few papers indeed indicate that both substrate preference and subcellular localization of some SULTR3s could be different than expected.

Kataoka et al. [31] reported that SULTR3;5 is expressed in the root vasculature of Arabidopsis— showing the same expression domain of the low-affinity SULTR2;1—and subcellular localizes on the

plasma membrane. The heterologous expression of *SULTR3;5* in yeasts defective for SO4 2− uptake shows that this protein does not transport SO4 2− itself, whereas it enhances the SO4 2− uptake capacity of SULTR2;1 when co-expressed in the same yeas<sup>t</sup> mutant. These results, along with the observation that the Arabidopsis *sultr3;5* mutant retains more SO4 2− in the root under S starvation, strongly sugges<sup>t</sup> that SULTR3;5 may have the function to help SULTR2;1 in retrieval apoplastic SO4 2−, contributing in this way to root-to-shoot SO4 2− translocation (Figure 1).

SULTR3;4 from rice and Arabidopsis have been recently indicated as SULTR-like phosphorus distribution transporters (SPDTs) playing essential roles in controlling the allocation of phosphate to grains and developing tissues, respectively [32,33]. Tissue-specific expression analyses show that SULTR3;4/SPDT of rice is expressed in the xylem region of both enlarged- and di ffuse-vascular bundles of nodes [32]. The Arabidopsis ortholog gene shows a more complex expression pattern, since it is mainly expressed in the fascicular cambium between the xylem and phloem and in the interfascicular cambium of lower stem, as well as in the cambial zone of the leaf petiole, rosette basal region, hypocotyl, and in the parenchyma cells of both xylem and phloem surrounding the cambial zone [33]. Moreover, the SULTR3;4/SPDTs are localized at the plasma membrane, show proton-dependent transport activities for phosphate, do not transport SO4 2−, and are up-regulated by phosphate deficiency but not under S starvation [32,33]. Mutations in *OsSULTR3;4*/*SPDT* alter the distribution of P in rice plants, decreasing both total P (−20%) and phytate (−30%) in the brown de-husked grains, without a ffecting yield, seed germination, and seedling vigor.

Another member of group 3, SULTR3;3, has been indicated as implicated in PA accumulation in barley and rice grains. Zhao et al. [34] recently reported that disruptions in rice *SULTR3;3* gene are the casual events of two interesting allelic mutations, previously described as *lpa-MH86-1* and *Os-lpa-Z9B-1*, since they produce grains with a reduced concentration of both PA and total P [35]. Tissue-specific expression analyses reveal that OsSULTR3;3 is expressed in the vascular bundles of shoots, leaves, flowers, and seeds, but not in the roots. This protein seems to be localized in the endoplasmic reticulum, when expressed in onion epidermal cells, and it does not show any transport activity for both SO4 2− and phosphate when heterologously expressed in yeas<sup>t</sup> mutant strains defective for SO4 2− or phosphate uptake, or *Xenopus* oocytes. However—as underlined by Zhao et al. [34]—the lack of transport activity for SO4 2− or phosphate in heterologous systems does not necessarily mean that OsSULTR3;3 does not have a role in SO4 2− of phosphate transport, since its activity may depend on other proteins or post-translational modifications not present in non-plant hosts. Moreover, *OsSULTR3;3* mutations affect the concentrations of total P and phosphate of both root and shoot—which result higher in the mutants than in the wild type—but also reduce the concentrations of SO4 2− in the same organs. Finally, transcriptional analyses performed on developing grains reveal that *OsSULTR3;3* disruptions are associated with significant changes in the transcript level of genes involved in S and P homeostasis, suggesting a possible role of this gene in the cross-talk between the two nutrients [34]. Interestingly, a single base pair substitution in the last exon of an ortholog gene of *OsSULTR3;3* (designed as *HvST*) has also been identified as the causal event for the *low phytic acid* phenotype of the *lpa1-1* barley mutants [36].

Taken as a whole, these findings strongly indicate that expression domains and subcellular localizations, as well as substrate preferences of the SULTR3 subfamily members are variable, and may depend on plant species, development stage, or experimental approaches used to study their functions. Further e fforts will be necessary to understand better whether this variability could play a role in the regulation of SO4 2− fluxes under di fferent environmental conditions, also concerning the level of other essential mineral nutrients.
