**3. Sulfur and Phosphorous Interplay**

Similar to S, P is also an essential macronutrient for plants. P is found as phosphate ester in the majority of the molecular constituents essential for plant cell functions, including nucleic acids, proteins, phospholipids, sugars, ATP, and NADPH. Important aspects related to P acquisition and

homeostasis in plants have been recently reviewed elsewhere [37–39]. Here, we mainly focus our attention on S and P interplay by analyzing specific aspects related to SO4 2− transport and distribution inside the plants.

Although it is clear that S or P deficiencies have diverse phenotypic e ffects on plant growth, development, and productivity, intriguing interconnected responses to the internal levels of these two nutrients have been described at metabolic and transcriptional levels, suggesting the existence of coordination between S and P homeostasis. Rouached [40] pointed out that deficiency or surplus of only one of the two nutrients often results in changes in the expression levels of genes specifically involved in controlling the homeostasis of the other nutrient and underlined as comparable molecular mechanisms regulate both SO4 2− and phosphate transport in plants.

At the metabolic level, one of the most evident relationships between S and P is linked to membrane composition. It is known that cells can replace sulfolipids by phospholipids under S starvation, as well as they are able to replace phospholipids by sulfolipids and/or galactolipids under P starvation [41–46]. In Arabidopsis, the synthesis of sulfolipids is catalyzed by two enzymes, SQD1 and SQD2, whose expressions are increased by P starvation [42,43]. Although lipid shifts could be interpreted as adaptive mechanisms for plant survival under di fferent nutrient availabilities, the physiological and biochemical consequences of phospholipids-sulfolipids substitutions on plant membrane functions are still unclear. Reprogramming membrane compositions under nutrient deficiency could have profound impacts on both S and P availability for plant metabolism. Moreover, recent studies have shown that the lipid environment and lipid-protein interactions may have crucial roles in modulating the functions as well as the conformational dynamics of membrane transporters [47].

Unfortunately, our basic knowledge about the interactions between P metabolism and SO4 2− transport is limited. A few papers show that P deficiency or perturbations in P metabolism may impact the SO4 2− allocation inside the plants. It has been reported that SO4 2− concentration increases in roots and decreases in shoots of Arabidopsis as a consequence of reduced phosphate availabilities in the growing medium [48]. Transcriptional analysis of the main *SULTR* genes implicated in long-distance SO4 2− transport reveals that, under P starvation, the transcript of *AtSULTR1;3* accumulates in both roots and shoots, whereas that of *AtSULTR2;1* weakly accumulates only in the roots. In the same conditions, *AtSQD1* transcript increases in both roots and shoots, indicating that adaptive modulations of SULTRs controlling the inter-organ distribution of SO4 2− are required for the replacement of phospholipid by sulfolipids induced by P starvation [48]. Most of these responses seem to be dependent on *PHR1* (*PHOSPHATE RESPONSE1*), a gene encoding a protein belonging to the MYB-CC family transcription factors involved in the activation of several phosphate starvation-induced genes (PSI) [38,49]. PHR1 binds to an imperfect palindromic motif, named P1BS, which is prevalent in the promoter of the PSI genes [49,50]. Interestingly *cis*-regulatory motifs for PHR1-dependent gene activation are also present in the promoters of both *AtSULTR1;3* and *AtSQD1* genes, whose expressions are coherently reduced in the Arabidopsis *phr1* mutant grown under P starvation [48,51,52]. Interestingly, other evidence indicates *PHR1* as the convergen<sup>t</sup> point for the cross-talk between P and other essential nutrients, such as zinc and iron [53,54]. *AtSULTR2;1*, is up-regulated by P starvation in a PHR1-independent manner, since *AtSULTR2;1* transcript further accumulates in *phr1* P deficient plants [48]. In this context, the observation that the expression of miR395—the microRNA that mainly controls the spatial expression of *SULTR2;1* in vascular tissues—is suppressed under P deficiency, allows us to speculate about the existence of an extra regulatory circuit which controls the inter-organ distribution of SO4 2− under P starvation [55]. In this circuit (Figure 1): (i) the suppression of miR395 should allow SULTR2;1 to control root-to-shoot SO4 2− translocation via the xylem route, as well as the source-to-sink SO4 2− re-allocation via the phloem; (ii) PHR1 activates the expression of SULTR1;3 increasing further the capacity of the plants to move SO4 2− from source to sink tissues. Unfortunately, no other evidence is available to support this extra regulatory circuit further and to fully appreciate its possible physiological impact on S metabolism in P deficient plants. Finally, the observation that Arabidopsis lines engineered for low PA content show alterations in SO4 2− distribution and changes in expression of some *SULTRs*

suggests the existence of another level of complexity in the cross-talk between S and P, which directly involves PA [56].

### **4. SULTRs as Novel Elements in the** *lpa* **Network**

As mentioned above, genetic lesions in some genes putatively involved in sulfate transport result in *lpa* phenotypes in rice and barley [32,34,36]. Interestingly, all the mutations described thus far affect putative SULTR genes belonging to the SULTR3 subfamily, which includes elements whose functions are still objects of debate. For a detailed description of the *SULTR3*/*lpa* alleles, readers are referred to Cominelli et al. [57].

Differently from other SULTRs, whose capability to transport SO4<sup>2</sup>− has mostly been proven using yeas<sup>t</sup> mutants as heterologous expression systems, the function of the SULTR3s as SO4<sup>2</sup>− transporters has only been hypothesized on the base of their sequence homologies with other SULTRs. Moreover, species-specific differences could explain the variability observed for the subcellular membrane localization of SULTR3 subfamily members.

Mineral nutrients required for plant growth are absorbed by the roots from the soil solution and then released to the xylem to be translocated to different tissues together with the transpiration flow. However, transpiration cannot be considered as the sole driving force for the root-to-shoot movement of nutrients, since developing organs such as new leaves and seeds are not photosynthetically active. Recently, nodes of gramineous plants have been identified as the main actors controlling nutrient delivery to developing tissues in a transpiration-independent way [58,59]. Several rice transporters involved in the intervascular transfer of nutrients from enlarged vascular bundles to diffuse vascular bundles of nodes seem to be essential to ensure this process [59]. Among these, *OsSULTR3;4*/*SPDT* has been indicated as pivotal in controlling phosphate delivery to developing tissues since it shows a proton-dependent transport activity for phosphate (but not for SO4<sup>2</sup>−), and it is highly expressed in the node 1 of rice at the reproductive stage [32]. Moreover, *OsSULTR3;4*/*SPDT* knockout mutants reduce P allocation to new leaves and grains, raveling the essential role of this transporter in switching phosphate toward developing leaves and grains.

The recent finding that the Arabidopsis *SULTR3;4* ortholog gene also controls xylem-to-phloem phosphate transfer, strongly suggests that sequence homology of SULTR3;4 with other SULTRs does not necessarily indicate that they share the same function [33]. All these observations not only show SULTR3;4s as phosphate transporters rather than as SO4<sup>2</sup>− transporters, but may also explain the role of these proteins in the *lpa* network.

If, on the one hand, the recent description of SULTR3;4/SPDTs as phosphate transporters seems to leave no room for doubt, on the other, assessment of the role of SULTR3;3s on P allocation still appears challenging. Rice SULTR3;3 is mainly expressed in vascular tissues and does not show any transport activity for SO4<sup>2</sup>− and phosphate [34]. Further studies are thus needed to uncover its function and subcellular localization. However, the lack of specific transport activity for SO4<sup>2</sup>− has also been indicated for the Arabidopsis SULTR3;5, which has been described as an essential component of the SO4<sup>2</sup>− transport system that facilitates the root-to-shoot SO4<sup>2</sup>− translocation in the vasculature [31]. Although the mechanisms controlling SO4<sup>2</sup>− allocation in rice are still known, it is possible to speculate that also *OsSULT3;3* could have a role in SO4<sup>2</sup>− partitioning among organs, by helping the activity of some other vascular transporter, or in SO4<sup>2</sup>− transport into the chloroplast, as recently suggested for its ortholog in Arabidopsis [30]. Rice *sultr3;3* mutants show significant alterations in S and P homeostasis, as indicated by the reduced concentration of SO4<sup>2</sup>− in both shoots and roots, as well as by the accumulation of transcripts of several S- and P-responsive genes in developing grains. Disruption in *OsSULTR3;3* also affects the concentrations of various grain metabolites not directly involved in PA biosynthesis. In particular, the reduced level of cysteine, along with the accumulation of its precursor serine, seems to indicate an insufficient supply of S during seed differentiation. Interestingly, reduced levels of cysteine have also been observed in the chloroplasts isolated from different Arabidopsis *sultr3* mutants [30]. Thus, the alterations in S homeostasis could be interpreted as the primary physiological

event that reduces the accumulation of P in the grains of *sultr3;3* mutants. Finally, since total P and phosphate concentrations in root and shoot are higher in mutants than in the wild type, we may further speculate about the existence of mechanisms that somehow limit the systemic mobility of P in the plant. The analysis of the membrane lipid composition could provide in the next future a possible explanation for this phenomenon since substitution of sulfolipids by phospholipids caused by an insu fficient S supply could increase the amount of P immobilized within cell membranes.
