Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.     

Impact of pedospheric and atmospheric sulphur nutrition on sulphur metabolism of Allium cepa L., a species with a potential sink capacity for secondary sulphur compounds

Onion (Allium cepa L.) was able to use atmospheric H(2)S as sole sulphur source for growth. The foliarly absorbed H(2)S was rapidly metabolized into water-soluble, non-protein thiol compounds, including cysteine, and subsequently into other sulphur compounds in the shoots. In H(2)S-exposed plants, the accumulation of sulphur compounds in the shoots was nearly linear with the concentration (0.15-0.6 microl l(-1)) and duration of the exposure. Exposure of onion to H(2)S for up to 1 week did not affect the sulphur content of the roots. Secondary sulphur compounds formed a sink for the foliarly absorbed sulphide, and the sulphur accumulation upon H(2)S exposure could, for a great part, be ascribed to enhancement of the content of gamma-glutamyl peptides and/or alliins. Furthermore, there was a substantial increase in the sulphate content in the shoots upon H(2)S exposure. The accumulation of sulphate originated both from the pedosphere and from the oxidation of absorbed atmospheric sulphide, and/or from the degradation of accumulated secondary sulphur compounds. From studies on the interaction between atmospheric and pedospheric sulphur nutrition it was evident that H(2)S exposure did not result in a down-regulation of the sulphate uptake by the roots.     

Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana

Adenosine 5'-phosphosulphate reductase (APR) is considered to be a key enzyme of sulphate assimilation in higher plants. We analysed the diurnal fluctuations of total APR activity and protein accumulation together with the mRNA levels of three APR isoforms of Arabidopsis thaliana. The APR activity reached maximum values 4 h after light onset in both shoots and roots; the minimum activity was detected at the beginning of the night. During prolonged light, the activity remained stable and low in shoots, but followed the normal rhythm in roots. On the other hand, the activity decreased rapidly to undetectable levels within 24 h of prolonged darkness both in shoots and roots. Subsequent re-illumination restored the activity to 50% in shoots and to 20% in roots within 8 h. The mRNA levels of all three APR isoforms showed a diurnal rhythm, with a maximum at 2 h after light onset. The variation of APR2 mRNA was more prominent compared to APR1 and APR3. 35SO42- feeding experiments showed that the incorporation of 35S into reduced sulphur compounds in vivo was significantly higher in light than in the dark. A strong increase of mRNA and protein accumulation as well as enzyme activity during the last 4 h of the dark period was observed, implying that light was not the only factor involved in APR regulation. Indeed, addition of 0.5% sucrose to the nutrient solution after 38 h of darkness led to a sevenfold increase of root APR activity over 6 h. We therefore conclude that changes in sugar concentrations are also involved in APR regulation.     

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.     

Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.     

Characterization of sulfate assimilation in marine algae focusing on the enzyme 5'-adenylylsulfate reductase

5'-Adenylylsulfate (APS) reductase was characterized in diverse marine algae. A cDNA encoding APS reductase from Enteromorpha intestinalis (EAPR) was cloned by functional complementation of an Escherichia coli cysH mutant. The deduced amino acid sequence shows high homology with APS reductase (APR) from flowering plants. Based on the probable transit peptide cleavage site the mature protein is 45.7 kD. EAPR expressed as a His-tagged recombinant protein catalyzes reduced glutathione-dependent reduction of APS to sulfite, exhibiting a specific activity of approximately 40 micromol min(-1) mg protein(-1) and Michealis-Menten kinetic constants of approximately 1.4 mM for reduced glutathione and approximately 6.5 microM for APS. APR activity and expression were studied in relation to the production of 3-dimethylsulfoniopropionate (DMSP), a sulfonium compound produced by many marine algae. A diverse group of DMSP-producing species showed extremely high enzyme activity (up to 400 times that found in flowering plants). Antibodies raised against a conserved peptide of APR strongly cross-reacted with a protein of 45 kD in several chlorophytes but insignificantly with chromophytes. In the chlorophyte Tetraselmis sp., APR activity varies significantly during the culture cycle and does not follow the changes in cellular DMSP content. However, a positive correlation was found between cell-based APR activity and specific growth rate.     

Atmospheric H(2)S as sulfur source for Brassica oleracea: kinetics of H(2)S uptake and activity of O-acetylserine (thiol)lyase as affected by sulfur nutrition

The uptake of hydrogen sulfide (H(2)S) by shoots of curly kale (Brassica oleracea) showed saturation kinetics with respect to the atmospheric concentration. The kinetics are largely determined by the rate of metabolism of the absorbed H(2)S into cysteine, catalyzed by O-acetylserine (thiol)lyase, and can be described by the Michaelis-Menten equation. When B. oleracea was grown under sulfate (SO(4)(2-))-deprived conditions, plants developed sulfur (S) deficiency symptoms and H(2)S uptake kinetics were substantially altered. Shoots of SO(4)(2-)-deprived plants had a lower affinity to H(2)S uptake, whereas the maximal H(2)S uptake rate was higher. When SO(4)(2-)-deprived plants were simultaneously exposed to 0.2 &mgr;l l(-1) H(2)S all S deficiency symptoms disappeared and H(2)S uptake kinetics returned rapidly to values observed for S-sufficient shoots. The activity of the H(2)S-fixating enzyme O-acetylserine (thiol)lyase was hardly affected upon either prolonged H(2)S exposure or SO(4)(2-) deprivation. Evidently, the activity of O-acetylserine (thiol)lyase was not the rate-limiting step in the H(2)S uptake by shoots. The significance of the in situ availability and rate of synthesis of the substrate O-acetylserine for O-acetylserine (thiol)lyase as determining factor in the uptake kinetics of H(2)S needs further evaluation.     

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.     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

Kinetic Assays for Determining In Vitro APS Reductase Activity in Plants without the Use of Radioactive Substances

Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.     

Genotypic variation in sulphur assimilation and metabolism of onion (Allium cepa L.). II: Characterisation of ATP sulphurylase activity

To investigate the regulation of sulphur (S)-assimilation in onion further at the biochemical level, the pungent cultivar W202A and the milder cultivar Texas Grano 438 PVP (TG) have been grown in S-sufficient (S⁺; 4 meq S⁻¹) or S-deficient (S⁻; 0.1 meq S⁻¹) growth conditions, and tissues excised at the seedling stage (pre-bulbing; ca. 10-weeks-old) and at the mature stage (bulbing; ca. 16-weeks-old). S-supply negatively influenced adenosine-5′-phosphosulphate (APS) reductase (APR) enzyme activity in both cultivars at bulbing only, and a higher abundance of APR was observed in both cultivars at bulbing in response to low S-supply. In contrast, S-supply significantly influenced ATP sulphurylase (ATPS) activity in leaf tissues of W202A only, and only at bulbing, while an increase in abundance in response to high S-supply was observed for both cultivars at bulbing. To investigate the regulation of the ATPS enzyme activity and accumulation further, activity was shown to decrease significantly in roots at bulbing in the S-deficient treatment in both cultivars, a difference that was only supported by western analyses in W202A. Phylogenetic analysis revealed that AcATPS1 groups in a broad monocot clade with the closest sequences identified in Sorghum bicolour, Zea mays and Oryza sativa, but with some support for a divergence of AcATPS1. Detection of ATPS in leaf extracts after two dimensional gel electrophoresis (2-DE) revealed that the protein may undergo post-translational modification with a differential pattern of ATPS accumulation detected in both cultivars over the developmental progression from the seedling to the bulbing stage. Treatment of leaf extracts of W202A to dephosphorylate proteins resulted in the loss of immuno-recognised ATPS spots after 2-DE separation, although enzyme activity was not influenced. These results are discussed in terms of the tiers of control that operate at the biochemical level in the reductive S-assimilation pathway in a S-accumulating species particularly during the high-S-demanding bulbing stage.     

Leaf age-dependent differences in sulphur assimilation and allocation in poplar (Populus tremula x P. alba) leaves

35S-sulphate was flap-fed to poplar leaves of different leaf development stages - young developing, expanding, mature, and old mature poplar leaves. (35)S-sulphate was taken up independent of the leaf development stage. Whereas young development leaves did not export the (35)S taken up, export increased with increasing leaf development stage. Expanding leaves allocated the exported (35)S mainly into apical tree parts (73-87%) and only to a minor extent (13-27%) in basipetal direction. Neither lower trunk sections nor the roots were sinks for the exported (35)S. Expanding and developing leaves, but not the shoot apex, were the main sinks for the (35)S allocated in apical direction. In contrast, mature and old mature leaves exported the (35)S taken up mainly in basipetal direction (65-82%) with the roots constituting the main sinks. The (35)S allocated into apical tree parts was found in expanding and developing leaves, but only to a minor extent in the shoot apex. Apical allocated (35)S was identified as sulphate. Apparently the demand of young developing leaves for reduced sulphur was not fulfilled by mature leaves. Therefore, reduced sulphur for growth and development of young developing leaves must be supplied from other sources. In vitro activity of enzymes involved in assimilatory sulphate reduction was measured to investigate whether demand for reduced sulphur by young leaves is met by their own sulphate reduction. ATP sulphurylase and APS reductase activities were not significantly lower in developing than in mature leaves. Sulphite reductase and serine acetyltransferase activities were highest in developing leaves; O:-acetylserine (thiol) lyase activity was similar in all leaf developing stages. Apparently, young developing poplar leaves are able to produce their own reduced sulphur for growth and development. Whether other sources such as storage tissues and/or roots are involved in reduced sulphur supply to developing leaves remains to be elucidated.     

Glutathione,a tripeptide which may function as a temporary storage compound of excessive reduced sulphur in H2S fumigated spinach plants

Summary Exposure of spinach plants to 250 ppb H₂S for two days resulted in a four-fold increase of the reduced glutathione (GSH)/sulphydryl (SH) concentration and in a two-fold increase of the oxidized glutathione (GSSG) concentration of the shoots. Both in the presence and the absence of H₂S, glutathione was predominantly present in the reduced form (more than 86%). When the H₂S exposure was ceased both the levels of GSH and GSSG in the shoot rapidly decreased. There was no emission of H₂S by the leaves after the fumigation was terminated. Glutathione reductase activity in the shoots was not affected by short term H₂S fumigation. It is proposed that glutathione plays the role of a temporary storage compound of excessive reduced sulphur in spinach shoots when exposed to H₂S in the ambient air.     

Copper toxicity in Chinese cabbage is not influenced by plant sulphur status,but affects sulphur metabolism‐related gene expression and the suggested regulatory metabolites

The toxicity of high copper (Cu) concentrations in the root environment of Chinese cabbage (Brassica pekinensis) was little influenced by the sulphur nutritional status of the plant. However, Cu toxicity removed the correlation between sulphur metabolism‐related gene expression and the suggested regulatory metabolites. At high tissue Cu levels, there was no relation between sulphur metabolite levels viz. total sulphur, sulphate and water‐soluble non‐protein thiols, and the expression and activity of sulphate transporters and expression of APS reductase under sulphate‐sufficient or‐deprived conditions, in the presence or absence of H₂S. This indicated that the regulatory signal transduction pathway of sulphate transporters was overruled or by‐passed upon exposure to elevated Cu concentrations.