Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.     

Phloem-localizing sulfate transporter,Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis. Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots. Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.     

植物硫转运蛋白研究进展

硫转运蛋白在植物对硫酸盐的吸收和转运中起着重要的作用。已经在拟南芥、大麦和小麦等植物中分离到了40多种硫转运蛋白基因。这些基因序列与其他种类生物的硫转运蛋白基因序列有着高度的保守性。利用CLUSTAL程序建立的系统进化树将植物硫转运蛋白划分为5个亚群。使用多种拓扑预测程序推测出不同植物硫转运蛋白的共同结构特点是均含有12个跨膜域。在柱花草和大麦中,硫转运蛋白基因表达调控包括植物体内硫水平的负调控和O—乙酰丝氨酸的正调控两种方式。对硫转运蛋白的组织定位和功能研究表明,高亲和硫转运蛋白主要定位于根部,在根系硫酸盐吸收中起重要作用。     

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M_r = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K_m for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

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.     

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.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Probing the function of STAS domains of the Arabidopsis sulfate transporters

Sulfate transporters in plants and animals are structurally conserved and have an amino-terminal domain that functions in transport and a carboxyl-terminal region that has been designated the STAS domain. The STAS domain in sulfate transporters has significant similarity to bacterial anti-sigma factor antagonists. To determine if the STAS domain has a role in controlling the activity of sulfate transporters, their stability, or their localization to the plasma membrane, we examined the effect of deleting or modifying the STAS domain of dominant sulfate transporters in roots of Arabidopsis thaliana. The A. thaliana Sultr1;2 and Sultr1;1 sulfate transporters rescue the methionine-dependent growth phenotype of the yeast sulfate transporter mutant strain CP154-7B. Constructs of Sultr1;2 in which the STAS domain was deleted (DeltaSTAS) resulted in synthesis of a truncated polypeptide that was unable to rescue the CP154-7B phenotype. The inability of these constructs to rescue the mutant phenotype probably reflected both low level cellular accumulation of the transporter and the inability of the truncated protein to localize to the plasma membrane. Fusing the STAS domain from other sulfate transporters to Sultr1;2 DeltaSTAS constructs restored elevated accumulation and plasma membrane localization, although the kinetics of sulfate uptake in the transformants were markedly altered with respect to transformants synthesizing wild-type Sultr1;2 protein. These results suggest that the STAS domain is essential, either directly or indirectly, for facilitating localization of the transporters to the plasma membrane, but it also appears to influence the kinetic properties of the catalytic domain of transporters.     

Cloning and characterization of two genes encoding sulfate transporters from rice (Oryza sativa L.)*

Two genes were isolated from a rice genomic library and the coding region of their corresponding cDNAs generated by RT-PCR. These single copy genes, designated ORYsa;Sultr1;1 and ORYsa;Sultr4;1, encode putative sulfate transporters. Both genes encode proteins with predicted topologies and signature sequences of the H⁺/SO₄^2- symporter family of transporters and exhibit a high degree of homology to other plant sulfate transporters. ORYsa;Sultr1;1 is expressed in roots with levels of expression being strongly enhanced by sulfate starvation. In situ hybridization experiments revealed that ORYsa;Sultr1;1 expression is localized to the main absorptive region of roots. This gene probably encodes a transporter that is responsible for uptake of sulfate from the soil solution. In contrast, ORYsa;Sultr4;1 was expressed in both roots and shoots and was unresponsive to the sulfur status of the plant. The sequence of ORYsa;Sultr4;1 contains a possible plastid-targeting transit peptide which may indicate a role in transport of sulfate to sites of sulfate reduction in plastids. The role of the transporter encoded by ORYsa;Sultr4;1 is likely to be significantly different fromORYsa;Sultr1;1. These are the first reports of isolation of genes encoding sulfate transporters from rice and provide a basis for further studies involving sulfate transport.     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

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.     

Coordinated expression of sulfate uptake and components of the sulfate assimilatory pathway in maize

A high-affinity-type sulfate transporter (Group 1: ZmST1;1, Accession No. AF355602) has been cloned from maize seedlings by RT-PCR. Tissue and cell specific localisation of this sulfate transporter has been determined along the developmental gradient of the root and in leaves of different ages. In S-sufficient conditions there was uniform low expression of ZmST1;1 in the root and very low expression in the leaves. Increased mRNA abundance and sulfate influx capacity indicated that S-starvation increased ZmST1;1 expression in roots, especially at the top of the root (just behind the seed, the area possessing most laterals and root hairs) compared to the root tip. Similarly a group 2, probable low affinity-type sulfate transporter, ZmST2;1, and also ATP-sulfurylase and APS-reductase but not OAS(thiol)lyase were induced by S-starvation and showed highest expression in the upper section of the root. S-starvation increased root/shoot ratio by 20 % and increased root lateral length and abundance in the region closest to the root tip. As the increase in root proliferation was not as great as the increase in mRNA pools, it was clear that there was a higher cellular abundance of the mRNAs for sulfate transporters, ATP-sulfurylase, and APS-reductase in response to sulfur starvation. In the leaves, the sulfate transporters, ATP-sulfurylase and APS-reductase were induced by S-starvation with the most mature leaf showing increased mRNA abundance first. In situ hybridization indicated that ZmST1;1 was expressed in epidermal and endodermal cell layers throughout the root whilst OAS(thiol)lyase was highly expressed in the root cortex.     

Transporter gene families in plants: the sulphate transporter gene family — redundancy or specialization?

Analysis of the Arabidopsis genome has indicated that the gene families encoding for specific solute transporters are surprisingly large. An example is the sulphate transporter family, which in Arabidopsis , consists of 14 isoforms showing homology to one another. H⁺-sulphate co-transport has been demonstrated for some, but not all, of these Arabidopsis isoforms. Occurrence of the sulphate transporter is ubiquitous and many examples have been cloned from a variety of plant species, animals and yeast. This is a unique transporter family with no apparent homology to any other transporter type. Phylogenetic analysis of the plant gene or amino acid sequences indicates that there are 5 or more distinguishable clusters within the family of sulphate transporters. Analysis of functional characteristics and patterns of regulation together with localization data, suggests that these groups have specific roles, such as high affinity uptake in the root, translocation in vascular tissues and cell to cell transfer in leaves and seeds. Additionally, some members of this sulphate transporter family may have discrete subcellular locations in plastid or tonoplast membranes. Within individual groups, there are often multiple isoforms from the same species, indicating an even greater degree of specialization or alternatively, redundancy. This mini-review will summarize the available data to distinguish between specialization and redundancy.     

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.     

The role of the STAS domain in the function and biogenesis of a sulfate transporter as probed by random mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.     

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K(m) = 0.1-0.3 mM), lower for Pro (K(m) = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K(m) = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.