Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions

Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 microM) or selenite (20 microM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mgkg(-1)) and three-fold in root Se concentration (up to 1000 mgkg(-1)). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mgkg(-1)) and two-fold in root (up to 650 mgkg(-1)). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F(1) offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F(1) offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon GxCol-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods.     

Global expression profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-acetyl-l-serine as a general regulator of gene expression in response to sulfur nutrition

To investigate the changes in profiles of mRNA accumulation in response to sulfur deficiency, approximately 13 000 non-redundant Arabidopsis thaliana ESTs corresponding to approximately 9000 genes were analyzed using DNA macroarray. Three-week-old Arabidopsis plants grown on an agarose-solidified control medium were transferred to a sulfate-free medium and grown for 48 h for the analyses of sulfur-related metabolites and global gene expression profiles. Concentrations of sulfate, O-acetyl-l-serine (OAS), a positive regulator of sulfur deficiency-responsive genes, cysteine and glutathione (GSH) were determined. Plants transferred to sulfate-free media had reduced concentrations of sulfate and GSH, and OAS concentrations increased. Macroarray analysis revealed a number of genes, including APR2 and Sultr1;2, whose mRNA accumulation was increased by sulfur deficiency. Profiling was also carried out with plants treated with OAS under sulfate-sufficient condition. Scatter plot analysis revealed a positive correlation between the changes of expression levels by sulfur deficiency and by OAS treatment among the clones tested, suggesting that mRNA accumulation of a number of genes under sulfur deficiency is mainly controlled by OAS concentrations in tissues. It was also revealed that the sets of genes regulated under sulfur deficiency in leaves and roots differ considerably.     

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.     

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5′ phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using ³⁴SO₄^2– also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non‐stress and oxidative stress conditions. However, when germinating on sulfur‐deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.     

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.     

Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium

Several Astragalus species have the ability to hyperaccumulate selenium (Se) when growing in their native habitat. Given that the biochemical properties of Se parallel those of sulfur (S), we examined the activity of key S assimilatory enzymes ATP sulfurylase (ATPS), APS reductase (APR), and serine acetyltransferase (SAT), as well as selenocysteine methyltransferase (SMT), in eight Astragalus species with varying abilities to accumulate Se. Se hyperaccumulation was found to positively correlate with shoot accumulation of S-methylcysteine (MeCys) and Se-methylselenocysteine (MeSeCys), in addition to the level of SMT enzymatic activity. However, no correlation was observed between Se hyperaccumulation and ATPS, APR, and SAT activities in shoot tissue. Transgenic Arabidopsis thaliana overexpressing both ATPS and APR had a significant enhancement of selenate reduction as a proportion of total Se, whereas SAT overexpression resulted in only a slight increase in selenate reduction to organic forms. In general, total Se accumulation in shoots was lower in the transgenic plants overexpressing ATPS, PaAPR, and SAT. Root growth was adversely affected by selenate treatment in both ATPS and SAT overexpressors and less so in the PaAPR transgenic plants. Such observations support our conclusions that ATPS and APR are major contributors of selenate reduction in planta. However, Se hyperaccumulation in Astragalus is not driven by an overall increase in the capacity of these enzymes, but rather by either an increased Se flux through the S assimilatory pathway, generated by the biosynthesis of the sink metabolites MeCys or MeSeCys, or through an as yet unidentified Se assimilation pathway.     

Sulfite oxidase controls sulfur metabolism under SO2 exposure in Arabidopsis thaliana

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range

Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored.Sulfur is one of the essential mineral nutrients for all organisms and is required for the biosynthesis of sulfur-containing amino acids, lipids, vitamins, and secondary metabolites, the catalytic and regulatory activity of enzymes, and the stabilization of protein structures. However, animals can only utilize sulfur that has been incorporated into biomolecules primarily originating from plants. Sufficient sulfur fertilization is also important to maximize yields of various crops, including rapeseed (Brassica napus) and wheat (Triticum aestivum; Bloem et al., 2004; Dubousset et al., 2010; Steinfurth et al., 2012). Interestingly, sulfur fertilization can also be important for food quality, with well-fertilized wheat crops producing flour with improved bread-making qualities (Shahsavani and Gholami, 2008).Over the last 50 years, the levels of sulfur in soils have been impacted significantly by the release of sulfur dioxide into the atmosphere as a result of the combustion of fossil fuels (Menz and Seip, 2004). In the atmosphere, sulfur dioxide is oxidized to sulfuric acid and thereafter deposited on soils as acid rain. Regulations controlling the release of sulfur dioxide from power stations introduced in the 1970s have caused significant declines in sulfate deposition due to this acid rain (Menz and Seip, 2004). These reductions in sulfate deposition are significant enough to have increased the need for sulfur fertilization of agricultural crops such as rapeseed and wheat (Dubousset et al., 2010; Steinfurth et al., 2012). The essentiality of sulfur for plants and the relatively recent major fluctuations in soil sulfate deposition make the study of natural variation in sulfur homeostasis by plants attractive. Such studies not only have the potential to identify new molecular mechanisms involved in sulfur homeostasis but also could provide a platform for probing at the genetic level interactions between natural plant populations and a changing soil environment.The sulfur analog selenium is also widely distributed in soil and is incorporated into biomolecules in plants in place of sulfur via sulfur assimilatory processes. Although selenium is not required by plants, it is essential for animals, and plant-based selenium is the primary source of this important nutrient for humans. In animals, selenium plays a vital role in numerous different selenoproteins involved in redox reactions, selenium storage, and hormone biosynthesis (Underwood, 1981; Rayman, 2012; Roman et al., 2014). In these proteins, selenium is incorporated as seleno-Cys. Unlike plants where selenium is incorporated into biomolecules nonspecifically as a sulfur analog, seleno-Cys in animals is biosynthesized by the action of a specific seleno-Cys synthase that converts Ser-tRNA to seleno-Cys-tRNA (Roman et al., 2014). To help prevent the negative health effects of selenium deficiency in the diet, fortification of various foods with selenium is already practiced, and biofortification of crops such as wheat, through the addition of selenium to fertilizers, is being proposed (Lyons et al., 2003). Moreover, there is a growing body of studies suggesting that supraoptimal dietary levels of selenium in the form of seleno-Met and seleno-methylseleno-Cys may be helpful in preventing certain cancers, although the efficacy of these supplements is still debated (Rayman, 2012; Steinfurth et al., 2012). Therefore, the identification of genes that control variation in the uptake and metabolism of sulfur and selenium in plants is not only an essential task for understanding the molecular mechanisms of plant nutrition but also important for crop yield, food quality, and human health.Plants mainly take up sulfur from the soil in the form of sulfate. After uptake of sulfate via the sulfate transporters SULTR1;1 and SULTR1;2 in the root (Yoshimoto et al., 2007; Barberon et al., 2008), sulfate needs to be reduced to sulfide before it can be incorporated into Cys, the first sulfur-containing compound in the assimilatory process (for review, see Takahashi et al., 2011). Sulfate is first activated through adenylation by ATP SULFURYLASE (ATPS) to form adenosine 5′-phosphosulfate (APS) in both plastids and the cytosol (Rotte and Leustek, 2000). After activation, sulfate as APS is reduced to sulfite by APS REDUCTASE (APR) using reduced glutathione (GSH) as the electron donor (Gutierrez-Marcos et al., 1996; Setya et al., 1996). Alternatively, APS can be phosphorylated by APS kinase to form 3′-phosphoadenosine 5′-phosphosulfate (PAPS), which acts as the sulfate donor for sulfotransferases to incorporate sulfate directly into saccharides and secondary metabolites such as glucosinolates (Mugford et al., 2009). Sulfite produced by APR is further reduced to sulfide by sulfite reductase (Khan et al., 2010). In the final step, sulfide is combined with O-acetyl-Ser to form Cys, catalyzed by O-acetyl-Ser(thiol)lyase (Wirtz and Hell, 2006). In plants, selenium is taken up as selenate via sulfate transporters (Shibagaki et al., 2002). After uptake, it is thought that selenate is reduced to selenite via the action of ATP sulfurylase and APS reductase (Shaw and Anderson, 1972; Pilon-Smits et al., 1999; Sors et al., 2005a, 2005b). Selenite is most likely nonenzymatically reduced to selenide and combined with O-acetyl-Ser to form seleno-Cys catalyzed by O-acetyl-Ser(thiol)lyase (Ng and Anderson, 1978).Sulfate uptake and reduction represent the two control points for sulfur homeostasis (Kopriva et al., 2009). Based on sequence similarity, there are 14 genes annotated as sulfate transporters in the Arabidopsis (Arabidopsis thaliana) genome, and at least six of them have evidence supporting their functions (Kopriva et al., 2009; Takahashi, 2010). Among these, SULTR1;1 and SULTR1;2 encode high-affinity sulfate transporters in the root responsible for sulfate uptake from the soil solution (Rouached et al., 2008; Takahashi, 2010). Genes encoding both ATPS and APR also form gene families in Arabidopsis, with ATPS being encoded by four genes and APR by three (Kopriva et al., 2009). ATPS enzymes localize to both the cytosol and plastids, whereas APR enzymes localize solely to the plastids (Lunn et al., 1990; Rotte and Leustek, 2000). ATPS1 and APR2 are responsible for the majority of ATP sulfurylase and APS reductase activity of seedlings.In a quantitative trait locus (QTL) analysis of sulfate content in Arabidopsis leaves using recombinant inbred lines generated by crossing Bayreuth (Bay-0) and Shahdara (Sha), APR2 was identified as a locus controlling variation in sulfate accumulation between the Bay-0 and Sha accessions (Loudet et al., 2007). The causal quantitative trait nucleotide in the Sha allele of APR2 results in a substitution of Ala-399 to Glu-399. The substitution localizes in the thioredoxin active site, which reduces the affinity of APR2 for GSH and results in a loss of 99.8% of enzyme activity. The loss-of-function Sha APR2 allele leads to a significant decrease in sulfate reduction and a concomitant increase in leaf sulfate accumulation. In the same analysis, a second QTL for leaf sulfate accumulation was described (Loudet et al., 2007). This second QTL was recently established to be driven by an ATPS1 expression-level polymorphism between Bay-0 and Sha, with Bay-0 showing reduced expression of ATPS1 compared with Sha (Koprivova et al., 2013).Both Loudet et al. (2007) and Koprivova et al. (2013) focused their studies on the genetic architecture of natural variation for leaf sulfate using a single recombinant population created by crossing the Bay-0 and Sha accessions. This recombinant inbred population has proved a powerful tool for the identification of novel alleles controlling several different traits (Loudet et al., 2008; Jiménez-Gómez et al., 2010; Jasinski et al., 2012; Pineau et al., 2012; Anwer et al., 2014). However, because this set of recombinant inbred lines is composed of genotypes from only two accessions, its value is limited when trying to understand allelic diversity across the Arabidopsis species as a whole. To address this limitation, we performed experiments using a set of 349 Arabidopsis accessions collected from across the species range. These 349 accessions were selected from a worldwide collection of 5,810 accessions sampled to minimize redundancy and close family relatedness (Baxter et al., 2010; Platt et al., 2010). To allow us to further probe Arabidopsis species-wide allelic diversity, we also utilized the complete genome sequences of 855 Arabidopsis accessions from the 1,001 Genomes Project (http://signal.salk.edu/atg1001/index.php).Since sulfate uptake and accumulation is only one step in the complex process of sulfur assimilation in plants, uncovering the genetic architecture of sulfate accumulation, as performed by Loudet et al. (2007) and Koprivova et al. (2013), could potentially overlook genetic variation in other important aspects of sulfur metabolism. To enable us to identify natural genetic variation in sulfur homeostasis beyond just sulfate accumulation, we chose to quantify total leaf sulfur. This potentially allowed us to capture variation in the accumulation of both sulfate and other important sulfur-containing metabolites such as GSH and glucosinolates.Furthermore, to test genetically the long-held assumption that selenium in plants is metabolized as a sulfur analog, we also phenotyped the same set of plants for total leaf selenium. Through a combination of high-throughput elemental analysis, linkage mapping, genetic and transgenic complementation, reciprocal grafting, and protein haplotype analysis, we have established that the natural variation in both leaf sulfur and selenium content in Arabidopsis is controlled by several rare APR2 variants across the whole of the Arabidopsis species.     

ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana

Zinc is an essential micronutrient for plants, but it is toxic in excess concentrations. In Arabidopsis, additional iron (Fe) can increase Zn tolerance. We isolated a mutant, zinc tolerance induced by iron 1, designated zir1, with a defect in Fe-mediated Zn tolerance. Using map-based cloning and genetic complementation, we identified that zir1 has a mutation of glutamate to lysine at position 385 on γ-glutamylcysteine synthetase (GSH1), the enzyme involved in glutathione biosynthesis. The zir1 mutant contains only 15% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by a specific inhibitor of GSH1, buthionine sulfoximine, resulted in loss of Fe-mediated Zn tolerance, which provides further evidence that glutathione plays an essential role in Fe-mediated Zn tolerance. Two glutathione-deficient mutant alleles of GSH1, pad2-1 and cad2-1, which contain 22% and 39%, respectively, of the wild-type glutathione level, revealed that a minimal glutathione level between 22 and 39% of the wild-type level is required for Fe-mediated Zn tolerance. Under excess Zn and Fe, the recovery of shoot Fe contents in pad2-1 and cad2-1 was lower than that of the wild type. However, the phytochelatin-deficient mutant cad1-3 showed normal Fe-mediated Zn tolerance. These results indicate a specific role of glutathione in Fe-mediated Zn tolerance. The induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a specific role in Fe-mediated Zn tolerance in Arabidopsis. We conclude that glutathione is required for the cross-homeostasis between Zn and Fe in Arabidopsis.     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

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.     

Enhanced glutathione metabolism is correlated with sulfur-induced resistance in Tobacco mosaic virus-infected genetically susceptible Nicotiana tabacum plants

Sulfur-induced resistance, also known as sulfur-enhanced defense (SIR/SED) was investigated in Nicotiana tabacum cv. Samsun nn during compatible interaction with Tobacco mosaic virus (TMV) in correlation with glutathione metabolism. To evaluate the influence of sulfur nutritional status on virus infection, tobacco plants were treated with nutrient solutions containing either sufficient sulfate (+S) or no sulfate (-S). Sufficient sulfate supply resulted in a suppressed and delayed symptom development and diminished virus accumulation over a period of 14 days after inoculation as compared with -S conditions. Expression of the defense marker gene PR-1a was markedly upregulated in sulfate-treated plants during the first day after TMV inoculation. The occurrence of SIR/SED correlated with a higher level of activity of sulfate assimilation, cysteine, and glutathione metabolism in plants treated with sulfate. Additionally, two key genes involved in cysteine and glutathione biosynthesis (encoding adenosine 5'-phosphosulfate reductase and γ-glutamylcysteine synthetase, respectively) were upregulated within the first day after TMV inoculation under +S conditions. Sulfate withdrawal from the soil was accelerated at the beginning of the infection, whereas it declined in the long term, leading to an accumulation of sulfur in the soil of plants grown with sulfate. This observation could be correlated with a decrease in sulfur contents in TMV-infected leaves in the long term. In summary, this is the first study that demonstrates a link between the activation of cysteine and glutathione metabolism and the induction of SIR/SED during a compatible plant-virus interaction in tobacco plants, indicating a general mechanism behind SIR/SED.     

Malformed selenoproteins are removed by the ubiquitin--proteasome pathway in Stanleya pinnata

Despite the widely accepted belief that selenium toxicity in plants is manifested by the misincorporation of selenocysteine into selenoproteins, there is a lack of data suggesting that selenoproteins are malformed or misfolded. Plant mechanisms to prevent the formation of selenoproteins are associated with increased selenium tolerance, yet there is no evidence to suggest that selenoproteins are malformed or potentially misfolded. We reasoned that if selenoproteins are malformed, then they might be degraded by the ubiquitin-proteasome pathway. The data demonstrate that selenate treatment induced the accumulation of both oxidized and ubiquitinated proteins, thus implicating both the 20S and 26S proteasome of Stanleya pinnata, a selenium-hyperaccumulating plant, in a selenate response. Inhibition of the proteasome increases the amount of selenium incorporated into protein, but not other elements. Furthermore, a higher percentage of selenium was found in a ubiquitinated protein fraction compared with other elements, suggesting that malformed selenoproteins are preferentially ubiquitinated and removed by the proteasome. Additionally, levels of the 20S and 26S proteasome and two heat shock proteins increase upon selenate treatment. Arabidopsis mutants with defects in the 26S proteasome have decreased selenium tolerance, which further supports the hypothesis that the 26S proteasome probably prevents selenium toxicity by removing selenoproteins.