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.     

Mechanisms of the selenium tolerance of the Arabidopsis thaliana knockout mutant of sulfate transporter SULTR1;2

We investigated the mechanism of selenium (Se) tolerance using an Arabidopsis thaliana knockout mutant of a sulfate transporter, sultr1;2. Se stress inhibited plant growth, decreased chlorophyll contents, and increased protein oxidation and lipid peroxidation in the wild type, whereas the sultr1;2 mutation mitigated damage of these forms, indicating that sultr1;2 is more tolerant of Se than the wild type is. The accumulation of symplastic Se was suppressed in sultr1;2 as compared to the wild type, and the chemical speciation of Se in the mutant was different from that in the wild type. Regardless of Se stress, the activities of ascorbate peroxidase, catalase, and peroxidase in the mutant were higher than in the wild type, while the activity of superoxide dismutase in the mutant was the same as in the wild type. These results suggest that the sultr1;2 mutation confers Se tolerance on Arabidopsis by decreasing symplastic Se and maintaining antioxidant enzyme activities.     

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.     

Glutathione homeostasis and Cd tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">sultr1;1</Emphasis>-<Emphasis Type="Italic">sultr1;2</Emphasis> double mutant with limiting sulfate supply

Key message

Cadmium sensitivity in sultr1;1 - sultr1;2 double mutant with limiting sulfate supply is attributed to the decreased glutathione content that affected oxidative defense but not phytochelatins’ synthesis.

Abstract

In plants, glutathione (GSH) homeostasis plays pivotal role in cadmium (Cd) detoxification. GSH is synthesized by sulfur (S) assimilation pathway. Many studies have tried to investigate the role of GSH homeostasis on Cd tolerance using mutants; however, most of them have focused on the last few steps of S assimilation. Until now, mutant evidence that explored the relationship between GSH homeostasis on Cd tolerance and S absorption is rare. To further reveal the role of GSH homeostasis on Cd stress, the wild-type and a sultr1;1-sultr1;2 double mutant which had a defect in two distinct high-affinity sulfate transporters were used in this study. Growth parameters, biochemical or zymological indexes and S assimilation-related genes’ expression were compared between the mutant and wild-type Arabidopsis plants. It was found that the mutations of SULTR1;1 and SULTR1;2 did not affect Cd accumulation. Compared to the wild-type, the double mutant was more sensitive to Cd under limited sulfate supply and suffered from stronger oxidative damage. More importantly, under the same condition, lower capacity of S assimilation resulted in decreased GSH content in mutant. Faced to the limited GSH accumulation, mutant seedlings consumed a large majority of GSH in pool for the synthesis of phytochelatins rather than participating in the antioxidative defense. Therefore, homeostasis of GSH, imbalance between antioxidative defense and severe oxidative damage led to hypersensitivity of double mutant to Cd under limited sulfate supply.

     

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.     

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.     

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.     

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.     

Nuclear Localised MORE SULPHUR ACCUMULATION1 Epigenetically Regulates Sulphur Homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S-adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over-accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation.     

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 .     

Overexpression of the sulfate transporter-encoding SULTR2 increases chromium accumulation in Chlamydomonas reinhardtii

Hexavalent chromium [Cr(Ⅵ)] is a highly toxic contaminant in aquatic systems, and microalgae represent promising bioremediators of metal-containing wastewater. However, the metal-binding capacity of algal cells is limited. Therefore, we improved the cellular Cr(Ⅵ) biosorption capacity of Chlamydomonas reinhardtii by overexpressing the sulfate transporter gene SULTR2. SULTR2 was predominantly located in the cytoplasm of the cell, and few proteins mobilized to the cell membrane as a Cr transporter under Cr stress conditions. Intracellular Cr accumulation was almost doubled in SULTR2-overexpressing transgenic strains after exposure to 30 μM K₂Cr₂O₇ for 4 d. Alginate-based immobilization increased the rate of Cr removal from 43.81% to 88.15% for SULTR2-overexpressing transgenic strains after exposure to 10 μM K₂Cr₂O₇ for 6 d. The immobilized cells also displayed a significant increase in nutrient removal efficiency compared to that of free-swimming cells. Therefore, SULTR2 overexpression in algae has a great potential for the bioremediation of Cr(Ⅵ)-containing wastewater.     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.