Regulation of high-affinity sulphate transporters in plants: towards systematic analysis of sulphur signalling and regulation

Plants require the function of plasma membrane-bound sulphate transporters for the initial uptake of inorganic sulphate. Part of this fundamental process is the energy-dependent proton/sulphate co-transport systems that are located in the surface cell layers of roots. During sulphur limitation, plants are able to activate the expression of sulphate transporters that facilitate the uptake of sulphate in roots. SULTR1;1 and SULTR1;2 are suggested to be the essential components of the sulphate uptake system in Arabidopsis roots. The physiological importance of SULTR1;1 and SULTR1;2 is supported by characteristics that can cope with sulphur deficiency: they were (i) functional high-affinity sulphate transporters; (ii) induced by sulphur limitation at the mRNA levels; and (iii) predominantly localized in the root hairs, epidermis, and cortex. The expression of high-affinity sulphate transporters was primarily regulated by sulphur in a promoter-dependent manner. Aside from the sulphur-specific regulation, the induction of SULTR1;1 and SULTR1;2 high-affinity sulphate transporters by sulphur limitation was dependent on the supply of carbon and nitrogen. In this review, the application of SULTR promoter-GFP systems for the analysis of regulatory pathways of sulphate acquisition in plants is described.     

SULTR3;1 is a chloroplast‐localized sulfate transporter in Arabidopsis thaliana

Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid‐localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1‐GFP localization, Western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss of SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S‐SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.     

Structural and functional analysis of the C-terminal STAS (sulfate transporter and anti-sigma antagonist) domain of the Arabidopsis thaliana sulfate transporter SULTR1.2

The C-terminal region of sulfate transporters from plants and animals belonging to the SLC26 family members shares a weak but significant similarity with the Bacillus sp. anti-anti-sigma protein SpoIIAA, thus defining the STAS domain (sulfate transporter and anti-sigma antagonist). The present study is a structure/function analysis of the STAS domain of SULTR1.2, an Arabidopsis thaliana sulfate transporter. A three-dimensional model of the SULTR1.2 STAS domain was built which indicated that it shares the SpoIIAA folds. Moreover, the phosphorylation site, which is necessary for SpoIIAA activity, is conserved in the SULTR1.2 STAS domain. The model was used to direct mutagenesis studies using a yeast mutant defective for sulfate transport. Truncation of the whole SULTR1.2 STAS domain resulted in the loss of sulfate transport function. Analyses of small deletions and mutations showed that the C-terminal tail of the SULTR1.2 STAS domain and particularly two cysteine residues plays an important role in sulfate transport by SULTR1.2. All the substitutions made at the putative phosphorylation site Thr-587 led to a complete loss of the sulfate transport function of SULTR1.2. The reduction or suppression of sulfate transport of the SULTR1.2 mutants in yeast was not due to an incorrect targeting to the plasma membrane. Both our three-dimensional modeling and mutational analyses strengthen the hypothesis that the SULTR1.2 STAS domain is involved in protein-protein interactions that could control sulfate transport.     

A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation

Cytokinin is an adenine derivative plant hormone that generally regulates plant cell division and differentiation in conjunction with auxin. We report that a major cue for the negative regulation of sulfur acquisition is executed by cytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor in Arabidopsis root. We constructed a green fluorescent protein (GFP) reporter system that generally displays the expression of the high-affinity sulfate transporter SULTR1;2 in Arabidopsis roots. GFP under the control of SULTR1;2 promoter showed typical sulfur responses that correlate with the changes in SULTR1;2 mRNA levels; accumulation of GFP was induced by sulfur limitation (-S), but was repressed in the presence of reduced sulfur compounds. Among the plant hormones tested, cytokinin significantly downregulated the expression of SULTR1;2. SULTR1;1 conducting sulfate uptake in sultr1;2 mutant was similarly downregulated by cytokinin. Downregulation of SULTR1;1 and SULTR1;2 by cytokinin correlated with the decrease in sulfate uptake activities in roots. The effect of cytokinin on sulfate uptake was moderated in the cre1-1 mutant, providing genetic evidence for involvement of CRE1/WOL/AHK4 in the negative regulation of high-affinity sulfate transporters. These data demonstrated the physiological importance of the cytokinin-dependent regulatory pathway in acquisition of sulfate in roots. Our results suggested that two different modes of regulation, represented as the -S induction and the cytokinin-dependent repression of sulfate transporters, independently control the uptake of sulfate in Arabidopsis roots.     

Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature

Xylem transport of sulfate regulates distribution of sulfur in vascular plants. Here, we describe SULTR3;5 as an essential component of the sulfate transport system that facilitates the root-to-shoot transport of sulfate in the vasculature. In Arabidopsis (Arabidopsis thaliana), SULTR3;5 was colocalized with the SULTR2;1 low-affinity sulfate transporter in xylem parenchyma and pericycle cells in roots. In a yeast (Saccharomyces cerevisiae) expression system, sulfate uptake was hardly detectable with SULTR3;5 expression alone; however, cells coexpressing both SULTR3;5 and SULTR2;1 showed substantial uptake activity that was considerably higher than with SULTR2;1 expression alone. The V(max) value of sulfate uptake activity with SULTR3;5-SULTR2;1 coexpression was approximately 3 times higher than with SULTR2;1 alone. In Arabidopsis, the root-to-shoot transport of sulfate was restricted in the sultr3;5 mutants, under conditions of high SULTR2;1 expression in the roots after sulfur limitation. These results suggested that SULTR3;5 is constitutively expressed in the root vasculature, but its function to reinforce the capacity of the SULTR2;1 low-affinity transporter is only essential when SULTR2;1 mRNA is induced by sulfur limitation. Consequently, coexpression of SULTR3;5 and SULTR2;1 provides maximum capacity of sulfate transport activity, which facilitates retrieval of apoplastic sulfate to the xylem parenchyma cells in the vasculature of Arabidopsis roots and may contribute to the root-to-shoot transport of sulfate.     

The function of SULTR2;1 sulfate transporter during seed development in Arabidopsis thaliana

SULTR2;1 is a low-affinity sulfate transporter expressed in the vascular tissues of roots and leaves for interorgan transport of sulfate in Arabidopsis thaliana . Transgenic Arabidopsis carrying a fusion gene construct of SULTR2;1 5'-promoter region and β-glucuronidase coding sequence (GUS) demonstrated that within the reproductive tissues, SULTR2;1 is specifically expressed in the bases and veins of siliques and in the funiculus, which connects the seeds and the silique. The antisense suppression of SULTR2;1 mRNA caused decrease of sulfate contents in seeds and of thiol contents both in seeds and leaves, as compared with the wildtype (WT). The effect of antisense suppression of SULTR2;1 on seed sulfur status was determined by introducing a sulfur-indicator construct, p35S::β_SRx3:GUS, which drives the expression of GUS reporter under a chimeric cauliflower mosaic virus 35S promoter containing a triplicate repeat of sulfur-responsive promoter region of soybean β-conglycinin β subunit (β_SRx3). The mature seeds of F1 plants carrying both the SULTR2;1 antisense and p35S::β_SRx3:GUS constructs exhibited significant accumulation of GUS activities on sulfur deficiency, as compared with those carrying only the p35S::β_SRx3:GUS construct in the WT background. These results suggested that SULTR2;1 is involved in controlling translocation of sulfate into developing siliques and may modulate the sulfur status of seeds in A. thaliana .     

Unequal functional redundancy between the two Arabidopsis thaliana high-affinity sulphate transporters SULTR1;1 and SULTR1;2

* In Arabidopsis, SULTR1;1 and SULTR1;2 are two genes proposed to be involved in high-affinity sulphate uptake from the soil solution. We address here the specific issue of their functional redundancy for the uptake of sulphate and for the accumulation of its toxic analogue selenate with regard to plant growth and selenate tolerance. * Using the complete set of genotypes, including the wild-type, each one of the single sultr1;1 and sultr1;2 mutants and the resulting double sultr1;1-sultr1;2 mutant, we performed a detailed phenotypic analysis of root length, shoot biomass, sulphate uptake, sulphate and selenate accumulation and selenate tolerance. * The results all ordered the four different genotypes according to the same functional hierarchy. Wild-type and sultr1;1 mutant plants displayed similar phenotypes. By contrast, sultr1;1-sultr1;2 double-mutant plants showed the most extreme phenotype and the sultr1;2 mutant displayed intermediate performances. Additionally, the degree of selenate tolerance was directly related to the seedling selenate content according to a single sigmoid regression curve common to all the genotypes. * The SULTR1;1 and SULTR1;2 genes display unequal functional redundancy, which leaves open for SULTR1;1 the possibility of displaying an additional function besides its role in sulphate membrane transport.     

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.     

Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [(35)S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [(35)S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.