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.     

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.     

Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity

APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).     

The role of the novel adenosine 5′-phosphosulfate reductase in regulation of sulfate assimilation of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Sulfate assimilation provides reduced sulfur for the synthesis of the amino acids cysteine and methionine and for a range of other metabolites. The key step in control of plant sulfate assimilation is the reduction of adenosine 5′-phosphosulfate to sulfite. The enzyme catalyzing this reaction, adenosine 5′phosphosulfate reductase (APR), is found as an iron sulfur protein in plants, algae, and many bacteria. In the moss Physcomitrella patens, however, a novel isoform of the enzyme, APR-B, has recently been discovered lacking the co-factor. To assess the function of the novel APR-B we used homologous recombination to disrupt the corresponding gene in P. patens. The knock-out plants were able to grow on sulfate as a sole sulfur source and the content of low molecular weight thiols was not different from wild type plants or plants where APR was disrupted. However, when treated with low concentrations of cadmium the APR-B knockout plants were more sensitive than both wild type and APR knockouts. In wild type P. patens, the two APR isoforms were not affected by treatments that strongly regulate this enzyme in flowering plants. The data thus suggest that in P. patens APS reduction is not the major control step of sulfate assimilation.     

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.     

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).     

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.     

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.     

The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae

We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite. To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation. Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile. Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator. The results obtained after the addition of DL-propargylglycine (5 microM and 100 microM) and cystathionine (50 microM) suggested that the intracellular cysteine level and cystathionine gamma-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation. Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.     

Influence of Chilling Stress on the Intercellular Distribution of Assimilatory Sulfate Reduction and Thiols in Zea mays

Abstract: The effect of chilling on the intercellular distribution of mRNAs for enzymes of assimilatory sulfate reduction, the activity of adenosine 5'-phosphosulfate reductase (APR), and the level of glutathione was analysed in leaves and roots of maize ( Zea mays L). At 25 °C the mRNAs for APR, ATP sulfurylase, and sulfite reductase accumulated in bundle-sheath only, whereas the mRNA for O-acetylserine sulfhydrylase was also detected in mesophyll cells. Glutathione was predominantly detected in mesophyll cells; however, oxidized glutathione was equally distributed between the two cell types. Chilling at 12 °C induced oxidative stress which resulted in increased concentrations of oxidized glutathione in both cell types and a prominent increase of APR mRNA and activity in bundle-sheath cells. After chilling, mRNAs for APR and sulfite reductase, as well as low APR activity, were detected in mesophyll cells. In roots, APR mRNA and activity were at higher levels in root tips than in the mature root and were greatly increased after chilling. These results demonstrate that chilling stress affected the levels and the intercellular distribution of mRNAs for enzymes of sulfate assimilation.     

Control of sulfur partitioning between primary and secondary metabolism

Sulfur is an essential nutrient for all organisms. Plants take up most sulfur as inorganic sulfate, reduce it and incorporate it into cysteine during primary sulfate assimilation. However, some of the sulfate is partitioned into the secondary metabolism to synthesize a variety of sulfated compounds. The two pathways of sulfate utilization branch after activation of sulfate to adenosine 5'-phosphosulfate (APS). Recently we showed that the enzyme APS kinase limits the availability of activated sulfate for the synthesis of sulfated secondary compounds in Arabidopsis. To further dissect the control of sulfur partitioning between the primary and secondary metabolism, we analysed plants in which activities of enzymes that use APS as a substrate were increased or reduced. Reduction in APS kinase activity led to reduced levels of glucosinolates as a major class of sulfated secondary metabolites and an increased concentration of thiols, products of primary reduction. However, over-expression of this gene does not affect the levels of glucosinolates. Over-expression of APS reductase had no effect on glucosinolate levels but did increase thiol levels, but neither glucosinolate nor thiol levels were affected in mutants lacking the APR2 isoform of this enzyme. Measuring the flux through sulfate assimilation using [(35) S]sulfate confirmed the larger flow of sulfur to primary assimilation when APS kinase activity was reduced. Thus, at least in Arabidopsis, the interplay between APS reductase and APS kinase is important for sulfur partitioning between the primary and secondary metabolism.     

The BIG gene is involved in regulation of sulfur deficiency-responsive genes in Arabidopsis thaliana

The Arabidopsis thaliana mutants altered sulfur response 1-1 ( asr1-1 ) and asr1-2 were isolated using the green fluorescent protein gene ( GFP ), as a marker, driven by a sulfur deficiency-responsive promoter containing the β_SR fragment, which is responsible for the induction of gene expression under sulfur deficiency. In the asr1 mutants, the expression of three sulfur deficiency-responsive genes β_SR-driven GFP , sulfate transporter 2;2 ( SULTR2;2 ) and adenosine-5'-phosphosulfate reductase 1 ( APR1 ) were induced in medium containing a normal sulfate concentration. The ASR1 locus was mapped to a 53-kb region on the upper arm of chromosome III; this is also the region of the BIG gene, which encodes a calossin-like protein necessary for the polar transport of auxin. The morphology of the asr1 mutants, i.e. reduced leaf size and inflorescence elongation, resembled that of big mutants. Using nucleotide sequence analysis of the BIG gene, we identified independent nonsense mutations in asr1-1 and asr1-2 . To confirm that ASR1 was BIG , we established lines of transgenic A. thaliana carrying a transfer DNA (T-DNA) insertion in the BIG gene. In these T-DNA insertion mutants, mRNA levels of β_SR-driven GFP and APR1 were upregulated in normal sulfate medium. The F₁ plants from crosses between asr1-1 and T-DNA insertion lines exhibited reduced leaf size and inflorescence length, indicating that ASR1 was indeed BIG . Taken together, the present results established that BIG is involved in the regulation of β_SR-driven GFP and APR1 mRNA level gene expression. Indole-3-acetic acid also upregulated β_SR-driven GFP and APR1 together with SULTR2;2 mRNA level, suggesting that the big effect on β_SR-driven GFP and APR1 is a pleiotropic aspect of the BIG gene.     

Control of sulphate assimilation and glutathione synthesis: interaction with N and C metabolism

Sulphate assimilation is an essential pathway being a source of reduced sulphur for various cellular processes and for the synthesis of glutathione, a major factor in plant stress defence. Many reports have shown that sulphate assimilation is well co-ordinated with the assimilation of nitrate and carbon. It has long been known that, during nitrate deficiency, sulphate assimilation is reduced and that the capacity to reduce nitrate is diminished in plants starved for sulphate. Only recently, however, was it shown that adenosine 5' phosphosulphate reductase (APR), the key enzyme of sulphate assimilation, is regulated by carbohydrates. In plants treated with sucrose or glucose APR was induced, whereas the activity was strongly reduced in plants grown in CO(2)-free air. The availability of cysteine is a crucial factor in glutathione synthesis, but an adequate supply of glutamate and glycine are also important. The molecular mechanisms for the co-ordination of S, N, and C assimilation are not known. O-acetylserine, a precursor of cysteine, was proposed to be the signal regulating sulphate assimilation, but most probably is not the outgoing signal to N and C metabolism. cDNA arrays revealed the induction of genes involved in auxin synthesis upon S-starvation, pointing to a possible role of phytohormones. Clearly, despite significant progress in understanding the regulation of sulphate assimilation and glutathione synthesis, their co-ordination with N and C metabolism achieved, and several potential signal molecules identified, present knowledge is still far from being sufficient.