High-affinity Sulfate Transporter Research Articles

Nitric oxide and hydrogen peroxide mediated regulation of chromium (VI) toxicity in wheat seedlings involves alterations in antioxidants and high affinity sulfate transporter

Chromium contamination of the soil is a major scientific concern with reference to crop productivity and human health. In recent years, several approaches are being employed in managing metal toxicity in crop plants. Here, we have investigated about potential and probable crosstalk of nitric oxide (NO) and hydrogen peroxide (H2O2) in mitigating hexavalent chromium [Cr(VI)] toxicity in wheat seedlings. Cr(VI) toxicity reduced the fresh mass and overall growth due to accumulation of reactive oxygen species (ROS) and decreased efficiency of AsA-GSH cycle and downregulation of high affinity sulfate transporter. However, exogenous treatment of NO and H2O2 significantly alleviated Cr toxicity. Application of NO and ROS scavengers reversed stress mitigating effects of NO and H2O2, respectively suggesting that endogenous NO and H2O2 are necessary for rendering Cr toxicity tolerance. Furthermore, NO rescued negative effect of diphenylene iodonium (DPI, NADPH oxidase inhibitor) and H2O2 reversed the negative effect of c-PTIO suggesting that they exhibit independent signalling in mitigating Cr stress. Altogether, data indicated that NO and H2O2 rendered mitigation of Cr stress by up-regulating enzymes (activity and relative gene expression) and metabolites of AsA-GSH cycle, high affinity sulfate transporter (relative gene expression) and glutathione biosynthesis which collectively controlled occurrence of oxidative stress.

Interaction between Sulfate and Selenate in Tetraploid Wheat (Triticum turgidum L.) Genotypes.

Selenium (Se) is an essential micronutrient of fundamental importance to human health and the main Se source is from plant-derived foods. Plants mainly take up Se as selenate (SeO42-), through the root sulfate transport system, because of their chemical similarity. The aims of this study were (1) to characterize the interaction between Se and S during the root uptake process, by measuring the expression of genes coding for high-affinity sulfate transporters and (2) to explore the possibility of increasing plant capability to take up Se by modulating S availability in the growth medium. We selected different tetraploid wheat genotypes as model plants, including a modern genotype, Svevo (Triticum turgidum ssp. durum), and three ancient Khorasan wheats, Kamut, Turanicum 21, and Etrusco (Triticum turgidum ssp. turanicum). The plants were cultivated hydroponically for 20 days in the presence of two sulfate levels, adequate (S = 1.2 mM) and limiting (L = 0.06 mM), and three selenate levels (0, 10, 50 μM). Our findings clearly showed the differential expression of genes encoding the two high-affinity transporters (TdSultr1.1 and TdSultr1.3), which are involved in the primary uptake of sulfate from the rhizosphere. Interestingly, Se accumulation in shoots was higher when S was limited in the nutrient solution.

Crosstalk Between Iron and Sulfur Homeostasis Networks in Arabidopsis.

The widespread deficiency of iron (Fe) and sulfur (S) is becoming a global concern. The underlying mechanisms regulating Fe and S sensing and signaling have not been well understood. We investigated the crosstalk between Fe and S using mutants impaired in Fe homeostasis, sulfate assimilation, and glutathione (GSH) biosynthesis. We showed that chlorosis symptoms induced by Fe deficiency were not directly related to the endogenous GSH levels. We found dynamic crosstalk between Fe and S networks and more interestingly observed that the upregulated expression of IRT1 and FRO2 under S deficiency in Col-0 was missing in the cad2-1 mutant background, which suggests that under S deficiency, the expression of IRT1 and FRO2 was directly or indirectly dependent on GSH. Interestingly, the bottleneck in sulfite reduction led to a constitutively higher IRT1 expression in the sir1-1 mutant. While the high-affinity sulfate transporter (Sultr1;2) was upregulated under Fe deficiency in the roots, the low-affinity sulfate transporters (Sultr2;1, and Sultr2;2) were down-regulated in the shoots of Col-0 seedlings. Moreover, the expression analysis of some of the key players in the Fe–S cluster assembly revealed that the expression of the so-called Fe donor in mitochondria (AtFH) and S mobilizer of group II cysteine desulfurase in plastids (AtNFS2) were upregulated under Fe deficiency in Col-0. Our qPCR data and ChIP-qPCR experiments suggested that the expression of AtFH is likely under the transcriptional regulation of the central transcription factor FIT.

Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation.

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2 S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2 S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H2 S and do not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2 S production as well as the potential use of these strains for winemaking and in yeast breeding programmes.

Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT.

A deficiency of the essential macronutrient sulfur leads to stunted plant growth and yield loss; however, an association with a symbiotic fungus can greatly improve nutrient uptake by the host plant. Here, we identified and functionally characterized a high-affinity sulfate transporter from the endophytic fungus Serendipita indica. SiSulT fulfills all the criteria expected of a functional sulfate transporter responding to sulfur limitation: SiSulT expression was induced when S. indica was grown under low-sulfate conditions, and heterologous expression of SiSulT complemented a yeast mutant lacking sulfate transport. We generated a knockdown strain of SiSulT by RNA interference to investigate the consequences of the partial loss of this transporter for the fungus and the host plant (maize, Zea mays) during colonization. Wild-type (WT) S. indica, but not the knockdown strain (kd-SiSulT), largely compensated for low-sulfate availability and supported plant growth. Colonization by WT S. indica also allowed maize roots to allocate precious resources away from sulfate assimilation under low-sulfur conditions, as evidenced by the reduction in expression of most sulfate assimilation genes. Our study illustrates the utility of the endophyte S. indica in sulfur nutrition research and offers potential avenues for agronomically sound amelioration of plant growth in low-sulfate environments.

High-Affinity Sulfate Transporter Sultr1;2 Is a Major Transporter for Cr(VI) Uptake in Plants.

Chromate (Cr[VI]) is a highly phytotoxic contaminant that is ubiquitous in soils. However, how Cr(VI) is taken up by plant roots remains largely unknown. Here, we show that the high-affinity sulfate transporter Sultr1;2 is responsible for Cr(VI) uptake by the roots of Arabidopsis thaliana. Sultr1;2 showed a much higher transport activity for Cr(VI) than Sultr1;1 when expressed in yeast cells. Knockdown of Sultr1;2 expression in Arabidopsis markedly reduced the Cr(VI) uptake rate, whereas knockout of Sultr1;1 had no or little effect. A double-knockout mutant (DKO) of the two genes lost the ability of Cr(VI) uptake almost completely. The Sultr1;2 knockdown mutant or DKO plants displayed higher resistance to Cr(VI) under normal sulfate conditions as a consequence of the lower tissue Cr accumulation. Overexpression of Sultr1;2 substantially increased Cr(VI) uptake with shoot Cr concentration being 1.6-2.0 times higher than that in the wild-type. These results indicate that Sultr1;2 is a major transporter responsible for Cr(VI) uptake in Arabidopsis, while Sultr1;1 plays a negligible role. Taken together, our study has identified a major transporter for Cr(VI) uptake in plants, providing potential strategies for engineering plants with low Cr accumulation and consequently enhanced Cr(VI) resistance and also plants with enhanced accumulation of Cr for the purpose of phytoremediation.

Regulation of Selenium/Sulfur Interactions to Enhance Chemopreventive Effects: Lessons to Learn from Brassicaceae.

Selenium (Se) is an essential trace element, which represents an integral part of glutathione peroxidase and other selenoproteins involved in the protection of cells against oxidative damage. Selenomethionine (SeMet), selenocysteine (SeCys), and methylselenocysteine (MeSeCys) are the forms of Se that occur in living systems. Se-containing compounds have been found to reduce carcinogenesis of animal models, and dietary supplemental Se might decrease cancer risk. Se is mainly taken up by plant roots in the form of selenate via high-affinity sulfate transporters. Consequently, owing to the chemical similarity between Se and sulfur (S), the availability of S plays a key role in Se accumulation owing to competition effects in absorption, translocation, and assimilation. Moreover, naturally occurring S-containing compounds have proven to exhibit anticancer potential, in addition to other bioactivities. Therefore, it is important to understand the interaction between Se and S, which depends on Se/S ratio in the plant or/and in the growth medium. Brassicaceae (also known as cabbage or mustard family) is an important family of flowering plants that are grown worldwide and have a vital role in agriculture and populations’ health. In this review we discuss the distribution and further interactions between S and Se in Brassicaceae and provide several examples of Se or Se/S biofortifications’ experiments in brassica vegetables that induced the chemopreventive effects of these crops by enhancing the production of Se- or/and S-containing natural compounds. Extensive further research is required to understand Se/S uptake, translocation, and assimilation and to investigate their potential role in producing anticancer drugs.

Tomato grafting onto Torubamu (Solanum melongena): miR166a and miR395b reduce scion Cd accumulation by regulating sulfur transport

This study aims to understand the mechanism by which grafting tomato (Solanum lycopersicum) onto Torubamu (Solanum melongena) reduces cadmium (Cd) accumulation in tomato. Tomato plants were grafted onto Torubamu rootstocks, and both the un-grafted and grafted tomato plants were grown in the control soil and soil that contained 2.0 or 5.0 mg•kg−1 Cd for 30 d. Grafting onto Torubamu rootstock markedly reduced the accumulation of both Cd and total sulfur in the leaves of tomato plants to 4.1–11.7% and 25.0–36.7% of the values in the un-grafted tomato plants, respectively. Moreover, the expression of miR166a and miR395b markedly increased in tomato scions. Tobacco dual-luciferase (LUC) assays and Agrobacterium-mediated tomato genetic transformation verified that miR166a and miR395b targeted Phavoluta (PHV), Revolute (REV), Class III Homeodomain Leucine Zipper (HD-ZIP), Sulfate transporter 2;1 (SULTR2;1), ATP sulfurylase (APS) (APS1/3), and High-affinity sulfate transporter (HAST) genes, which are directly or indirectly responsible for sulfate transport. Based on the correlations between the Cd and sulfur accumulation in the tomato scions (r2 = 0.91) and in the miR166a/395b-transformed tomato scions (r2 = 0.96–0.98) grown in the soil with 2.0 mg•kg−1 Cd, we conclude that tomato grafting onto Torubamu rootstock can reduce Cd accumulation because of the sulfate transport regulatory ability of miR166a and miR395b. Understanding this mechanism can be of great benefit for Cd tolerance breeding.

Contribution of Root Hair Development to Sulfate Uptake in Arabidopsis.

Root hairs often contribute to nutrient uptake from environments, but the contribution varies among nutrients. In Arabidopsis, two high-affinity sulfate transporters, SULTR1;1 and SULTR1;2, are responsible for sulfate uptake by roots. Their increased expression under sulfur deficiency (−S) stimulates sulfate uptake. Inspired by the higher and lower expression, respectively, of SULTR1;1 in mutants with more (werwolf [wer]) and fewer (caprice [cpc]) root hairs, we examined the contribution of root hairs to sulfate uptake. Sulfate uptake rates were similar among plant lines under both sulfur sufficiency (+S) and −S. Under −S, the expression of SULTR1;1 and SULTR1;2 was negatively correlated with the number of root hairs. These results suggest that both −S-induced SULTR expression and sulfate uptake rates were independent of the number of root hairs. In addition, we observed (1) a negative correlation between primary root lengths and number of root hairs and (2) a greater number of root hairs under −S than under +S. These observations suggested that under both +S and −S, sulfate uptake was influenced by the root biomass rather than the number of root hairs.

Chromium detoxification in arbuscular mycorrhizal symbiosis mediated by sulfur uptake and metabolism

It has been well demonstrated that arbuscular mycorrhizal (AM) symbiosis can improve plant chromium (Cr) tolerance, but the detoxification mechanisms are still unsolved. In the present study the role of AM symbiosis in plant sulfur (S) acquisition and metabolism related to Cr detoxification was investigated. Medicago truncatula plants were cultivated with/without AM fungus Rhizophagus irregularis in Cr(VI) contaminated soils at two soil S and phosphorus (P) concentrations. Sulfur uptake, transport and metabolism by plants were determined by biochemical and molecular methods. Meanwhile, Cr uptake and transformation were characterized by using spectroscopic methods, including synchrotron based X-ray absorption near edge structure (XANES) analysis. The results indicated that mycorrhizal inoculation improved plant S acquisition by up-regulating the expression of high affinity sulfate transporter genes (i.e. MtSULTR1.1, MtSULTR1.2) in plant roots. Mycorrhizal colonization also systematically regulated S transport and metabolism for Cr(VI) detoxification. XANES analysis revealed the Cr immobilization in AM roots by phosphate and histidine-like ligands rather than thiol groups. Correlation analysis further confirmed possible involvement of S metabolites in the relief of Cr induced oxidative stress. Mycorrhizal effects were more pronounced under low P condition. The study allowed a new insight into the mechanisms of enhanced plant Cr tolerance by AM symbiosis.

Molecular regulation of aluminum resistance and sulfur nutrition during root growth

Aluminum toxicity and sulfate deprivation both regulate microRNA395 expression, repressing its low-affinity sulfate transporter ( SULTR2;1 ) target. Sulfate deprivation also induces the high-affinity sulfate transporter gene ( SULTR12 ), allowing enhanced sulfate uptake. Few studies about the relationships between sulfate, a plant nutrient, and aluminum, a toxic ion, are available; hence, the molecular and physiological processes underpinning this interaction are poorly understood. The Al-sulfate interaction occurs in acidic soils, whereby relatively high concentrations of trivalent toxic aluminum (Al3+) may hamper root growth, limiting uptake of nutrients, including sulfur (S). On the other side, Al3+ may be detoxified by complexation with sulfate in the acid soil solution as well as in the root-cell vacuoles. In this review, we focus on recent insights into the mechanisms governing plant responses to Al toxicity and its relationship with sulfur nutrition, emphasizing the role of phytohormones, microRNAs, and ion transporters in higher plants. It is known that Al3+ disturbs gene expression and enzymes involved in biosynthesis of S-containing cysteine in root cells. On the other hand, Al3+ may induce ethylene biosynthesis, enhance reactive oxygen species production, alter phytohormone transport, trigger root growth inhibition and promote sulfate uptake under S deficiency. MicroRNA395, regulated by both Al toxicity and sulfate deprivation, represses its low-affinity Sulfate Transporter 2;1 (SULTR2;1) target. In addition, sulfate deprivation induces High Affinity Sulfate Transporters (HAST; SULTR1;2), improving sulfate uptake from low-sulfate soil solutions. Identification of new microRNAs and cloning of their target genes are necessary for a better understanding of the role of molecular regulation of plant resistance to Al stress and sulfate deprivation.

The Sulfate Supply Maximizing Arabidopsis Shoot Growth Is Higher under Long- than Short-Term Exposure to Cadmium.

The processes involved in cadmium detoxification in plants deeply affect sulfate uptake and thiol homeostasis and generate increases in the plant nutritional request for sulfur. Here, we present an analysis of the dependence of Arabidopsis growth on the concentration of sulfate in the growing medium with the aim of providing evidence on how plants optimize growth at a given sulfate availability. Results revealed that short-term (72 h) exposure to a broad range of Cd concentrations (0.1, 1, and 10 μM) inhibited plant growth but did not produce any significant effects on the growth pattern of both shoots and roots in relation to the external sulfate. Conversely, long-term (22 days) exposure to 0.1 μM Cd significantly changed the pattern of fresh weight accumulation of the shoots in relation to the external sulfate, without affecting that of the roots, although their growth was severely inhibited by Cd. Moreover, under long-term exposure to Cd, increasing the sulfate external concentration up to the critical value progressively reduced the inhibitory effects exerted by Cd on shoot growth, indicating the existence of sulfate-dependent adaptive responses protecting the shoot tissues against Cd injury. Transcriptional induction of the high-affinity sulfate transporter genes (SULTR1; 1 and SULTR1; 2) involved in sulfate uptake by roots was a common adaptive response to both short- and long-term exposure to Cd. Such a response was closely related to the total amount of non-protein thiols accumulated by a single plant under short-term exposure to Cd, but did not showed any clear relation with thiols under long-term exposure to Cd. In this last condition, Cd exposure did not change the level of non-protein thiols per plant and thus did not alter the nutritional need for sulfur. In conclusion, our results indicate that long term-exposure to Cd, although it induces sulfate uptake, decreases the capacity of the Arabidopsis roots to efficiently absorb the sulfate ions available in the growing medium making the adaptive response of SULTR1; 1 and SULTR1; 2 “per se” not enough to optimize the growth at sulfate external concentrations lower than the critical value.

Differential paralog divergence modulates genome evolution across yeast species.

Evolutionary outcomes depend not only on the selective forces acting upon a species, but also on the genetic background. However, large timescales and uncertain historical selection pressures can make it difficult to discern such important background differences between species. Experimental evolution is one tool to compare evolutionary potential of known genotypes in a controlled environment. Here we utilized a highly reproducible evolutionary adaptation in Saccharomyces cerevisiae to investigate whether experimental evolution of other yeast species would select for similar adaptive mutations. We evolved populations of S. cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. uvarum and S. cerevisiae for ~200–500 generations in sulfate-limited continuous culture. Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter gene, SUL1. However, while amplification of the SUL1 locus was detected in S. paradoxus and S. mikatae populations, S. uvarum cultures instead selected for amplification of the paralog, SUL2. We measured the relative fitness of strains bearing deletions and amplifications of both SUL genes from different species, confirming that, converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric promoter-ORF constructs, we were able to delineate the cause of this differential fitness effect primarily to the promoter of S. uvarum SUL1. Our data show evidence of differential sub-functionalization among the sulfate transporters across Saccharomyces species through recent changes in noncoding sequence. Furthermore, these results show a clear example of how such background differences due to paralog divergence can drive changes in genome evolution.

5'-non-transcribed flanking region and 5'-untranslated region play distinctive roles in sulfur deficiency induced expression of SULFATE TRANSPORTER 1;2 in Arabidopsis roots.

Plants increase sulfate uptake activity under sulfur deficiency (-S). In Arabidopsis, SULTR1;2 is the major high-affinity sulfate transporter induced in epidermis and cortex of roots for mediating sulfate uptake under -S. Though it is known that transcript levels of SULTR1;2 increase under -S largely due to the function of 5'-upstream region, contributions of 5'-non-transcribed flanking region and 5'-untranslated region (UTR) to transcriptional and post-transcriptional regulations have not yet been individually verified. To investigate the roles of 5'UTR of SULTR1;2 in -S responses, transcript levels and activities of firefly luciferase (Luc) were analyzed in transgenic plants expressing Luc under the control of the 2,160-bp long 5'-upstream region of SULTR1;2 with (PL2160) or without (PL2160ΔUTR) the 154-bp 5'UTR. Both transgenic plants expressed similar levels of Luc mRNAs that showed significant accumulations under -S relative to +S regardless of presence of the 5'UTR. In contrast, Luc activities were detected only in PL2160 plants, suggesting presence of 5'UTR of SULTR1;2 being necessary for translational initiation while its absence impairing translation of functional Luc protein in PL2160ΔUTR. These results indicate an essential role of the 5'-non-transcribed flanking region of SULTR1;2 at positions -2160 to -155 in -S-responsive transcriptional regulation.

High-Throughput Identification of Adaptive Mutations in Experimentally Evolved Yeast Populations.

High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in Saccharomyces cerevisiae using sets of single-gene deletions and amplifications of almost all the genes in the S. cerevisiae genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously—but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution.

Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis.

Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil –SO42−– is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco.

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach.

The effect of excess sulfate supply on iron accumulation in three graminaceous plants at the early vegetative phase

In recent years it has been established a significant relationship between sulfur (S) and iron (Fe) nutrition. In particular, it has been demonstrated that S deprivation can hinder Fe acquisition in barley, maize and wheat. This can be explained by assuming that, in order to cope with low Fe availability in the soil, grasses have evolved a phytosiderophores (PS)-based Fe chelation system (Strategy II) and PS are synthesized from methionine, through a nicotianamine intermediate.On the other hand, it has been demonstrated that wheat plants exhibit a higher Fe accumulation when supplied with excess S concentration, this effect being especially beneficial under severely limited Fe supply.The goal of this study was to explore whether the higher ability to acquire Fe, induced by a higher S supply, might be a general response of graminaceous species.The response of durum wheat (Triticum durum L.), barley (Hordeum vulgare L.), and maize (Zea mays L.) to the excess of S availability (2.4mM vs 1.2mM which is considered as optimal) was studied as a function of Fe availability (limited and sufficient, 20 and 80μM, respectively). At the end of the experimental period, which lasted 11 days, growth parameters (shoot and root fresh weight and chlorophyll content), total S and Fe concentrations, and PS release rate were compared among the three species. Furthermore, we evaluated plant sulfate uptake capability, by analysing the expression of genes coding for high affinity sulfate transporter (TdST1.1, HvST1.1 and ZmST1.1) in roots of each graminaceous plant.Our preliminary findings are largely consistent with the apparent divergence among the three species. In particular, an excess S supply may result in the improvement of Fe use efficiency in durum wheat plants, but not in both barley and maize.The use of higher S supply seems to be a promising approach, at least for wheat plants, which can both reduce agricultural demand for Fe fertilizers and improve the Fe use efficiency of plants.

Combinatorial use of sulfur-responsive regions of sulfate transporters provides a highly sensitive plant-based system for detecting selenate and chromate in the environment

ABSTRACTSelenium and chromium are heavy metals that are highly toxic to living organisms when they are present in excess amounts. The major forms of selenium and chromium in the environment are selenate and chromate, which act as toxic analogs of sulfate in plants, inhibiting its uptake and causing sulfur deficiency. To provide simple and inexpensive means of environmental analysis for selenate and chromate, we previously developed a model system for detecting and quantifying their levels by measuring the accumulation of green fluorescent protein (GFP) in transgenic plants under the control of the sulfur-responsive promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis (PSULTR1;2-GFP). However, the detection limit for both selenate and chromate was 10 μmol L−1, which was higher than the environmental standards. Here, we report a more sensitive monitoring system for selenium and chromium by combining PSULTR1;2 with another sulfur-responsive region of the sulfate transporter SULTR2;1 located in the 3ʹ-non-transcribed intergenic region (TSULTR2;1). The fusion gene construct consisting of PSULTR1;2, GFP and TSULTR2;1 (PSULTR1;2-GFP-TSULTR2;1) was introduced into Arabidopsis plants, and PSULTR1;2-GFP-TSULTR2;1 transgenic plants showed significant increases in GFP under sulfur-deficient conditions or with the addition of selenate or chromate to the growth medium. The increase in GFP depended on selenate concentration. Overall, the use of PSULTR1;2-GFP-TSULTR2;1 plants decreased the detection limits to 1 and 3 μmol L−1 for selenate and chromate, respectively, providing a highly sensitive plant-based system for detecting selenium and chromium in the environment.

Glutathione homeostasis and Cd tolerance in the Arabidopsis sultr1;1-sultr1;2 double mutant with limiting sulfate supply.

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. 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.