Regulation of sulfate assimilation by nitrogen and sulfur nutrition in poplar trees

Plants cover their need for sulfur by taking up inorganic sulfate, reducing it to sulfide, and incorporating it into the amino acid cysteine. In herbaceous plants the pathway of assimilatory sulfate reduction is highly regulated by the availability of the nutrients sulfate and nitrate. To investigate the regulation of sulfate assimilation in deciduous trees we used the poplar hybrid Populus tremula × P. alba as a model. The enzymes of the pathway are present in several isoforms, except for sulfite reductase and -glutamylcysteine synthetase; the genomic organization of the pathway is thus similar to herbaceous plants. The mRNA level of APS reductase, the key enzyme of the pathway, was induced by 3 days of sulfur deficiency and reduced by nitrogen deficiency in the roots, whereas in the leaves it was affected only by the withdrawal of nitrogen. When both nutrients were absent, the mRNA levels did not differ from those in control plants. Four weeks of sulfur deficiency did not affect growth of the poplar plants, but the content of glutathione, the most abundant low molecular thiol, was reduced compared to control plants. Sulfur limitation resulted in an increase in mRNA levels of ATP sulfurylase, APS reductase, and sulfite reductase, probably as an adaptation mechanism to increase the efficiency of the sulfate assimilation pathway. Altogether, although distinct differences were found, e.g. no effect of sulfate deficiency on APR in poplar leaves, the regulation of sulfate assimilation by nutrient availability observed in poplar was similar to the regulation described for herbaceous plants.     

Sulfate assimilation and glutathione synthesis in C<Subscript>4</Subscript> plants

Sulfate assimilation and glutathione synthesis were traditionally believed to be differentially compartmentalised in C₄ plants with the synthesis of cysteine and glutathione restricted to bundle sheath and mesophyll cells, respectively. Recent studies, however, showed that although ATP sulfurylase and adenosine 5′ phosphosulfate reductase, the key enzymes of sulfate assimilation, are localised exclusively in bundle sheath in maize and other C₄ monocot species, this is not true for the dicot C₄ species of Flaveria. On the other hand, enzymes of glutathione biosynthesis were demonstrated to be active in both types of maize cells. Therefore, in this review the recent findings on compartmentation of sulfate assimilation and glutathione metabolism in C₄ plants will be summarised and the consequences for our understanding of sulfate metabolism and C₄ photosynthesis will be discussed.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation

Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50 μM) and sulfur-sufficient (1,000 μM) conditions for 30 days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions.     

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.     

Kinetic mechanism of the dimeric ATP sulfurylase from plants

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PP_i (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PP_i in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.     

Heavy metal stress and sulfate uptake in maize roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.     

Influence of age and sulfur metabolism on ATP sulfurylase activity in the soybean and a survey of selected species

ATP sulfurylase activity varied greatly among different leaves on the soybean plant [Glycine max (L.) Meer.], and high levels of activity did not appear in the leaves until the seedlings were about 3 weeks old. In general, leaves from the top of the plant had a higher activity than leaves from the bottom of the plant. A much greater activity was found in soybean leaves than in soybean roots. The absence of sulfate in the nutrient solution resulted in higher enzyme activity in leaves from young plants and in lower activity in leaves from older plants. Over the growing season, however, ATP sulfurylase activity appeared to be related to sulfur content of the leaf. Several other plant species also had measurable levels of ATP sulfurylase.     

Sulfate Assimilation in C(4) Plants: Intercellular and Intracellular Location of ATP Sulfurylase, Cysteine Synthase, and Cystathionine beta-Lyase in Maize Leaves

The activity of ATP sulfurylase, cysteine synthase, and cystathionine β-lyase was measured in crude leaf extracts, bundle sheath strands, and mesophyll and bundle sheath chloroplasts to determine the location of sulfate assimilation of C₄ plant leaves. Almost all the ATP sulfurylase activity was located in the bundle sheath chloroplasts while cysteine synthase and cystathionine β-lyase activity was located, in different proportions, in both chloroplast types.

A new spectrophotometric assay for measuring ATP sulfurylase activity is also described.

     

Assimilatory sulfate reduction in C(3), C(3)-C(4), and C(4) species of Flaveria

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C(4) monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C(4) photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C(3), C(3)-C(4), C(4)-like, and C(4) species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C(4)-like and C(4) species than in C(3) and C(3)-C(4) species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C(4) species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C(4) plants and therefore is neither a prerequisite nor a consequence of C(4) photosynthesis.     

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.     

Interaction of sulfate assimilation with nitrate assimilation as a function of nutrient status and enzymatic co-regulation inbrassica juncea roots

The interaction of sulfate assimilation with nitrate assimilation inBrassica juncea roots was analyzed by monitoring the regulation of ATP sulfurylase (AS), adenosine-5’-phosphosulfate reductase (AR), sulfite reductase (SiR), and nitrite reductase (NiR). Depending on the status of sulfur and nitrogen nutrition, AS and AR activities and mRNA levels were increased by sulfate starvation but decreased by nitrate starvation. The activation of AS and AR by sulfate starvation was inhibited by sulfate/nitrate starvation. However, the rise in steady-state mRNA levels for AS and AR by sulfate starvation was not affected by sulfate/nitrate starvation. SiR gene expression was slightly activated by both sulfate starvation and sulfate/nitrate starvation, but was decreased by nitrate starvation. Although NiR gene expression was little affected by sulfate starvation, it was diminished significantly by either nitrate or nitrate/sulfate starvation. Cysteine (Cys) also decreased AS and AR activities and mRNA levels even when plants were simultaneously starved for sulfate; in contrast, both SiR and NiR gene expressions were only slightly, if at all, affected under the same conditions. This supports our conclusion that Cys, the end-product of sulfate assimilation, is the key regulatory signal. Moreover, SiR and NiR apparently are not the linking step in the co-regulation of sulfate and nitrate assimilation in plants.     

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.     

嗜酸氧化亚铁硫杆菌ATP硫酸化酶的表达、纯化及性质鉴定

ATP硫酸化酶是一种催化ATP和SO42-反应生成腺嘌呤-5’-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,它是硫酸根同化反应第一步的关键酶。以嗜酸氧化亚铁硫杆菌(A.ferrooxidansATCC 23270)基因组为模板,用PCR扩增得到ATPS基因,并克隆到表达载体pLM1上。加入IPTG的诱导表达,用AKTA蛋白纯化仪的镍柱亲和层析纯化得到浓度和纯度都较高的ATPS蛋白。SDS-PAGE分析,证实其分子量大小为33 kD,并成功的测出了其活性,比活达3.0×103U/mg。     

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants

Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).