Sulfate assimilation in basal land plants - what does genomic sequencing tell us?

Sulfate assimilation is a pathway providing reduced sulfur for the synthesis of cysteine, methionine, co-enzymes such as iron-sulfur centres, thiamine, lipoic acid, or Coenzyme A, and many secondary metabolites, e.g., glucosinolates or alliins. The pathway is relatively well understood in flowering plants, but very little information exists on sulfate assimilation in basal land plants. Since the finding of a putative 3'-phosphoadenosine 5'-phosphosulfate reductase in PHYSCOMITRELLA PATENS, an enigmatic enzyme thought to exist in fungi and some bacteria only, it has been evident that sulfur metabolism in lower plants may substantially differ from seed plant models. The genomic sequencing of two basal plant species, the Bryophyte PHYSCOMITRELLA PATENS, and the Lycophyte SELAGINELLA MOELLENDORFFII, opens up the possibility to search for differences between lower and higher plants at the genomic level. Here we describe the similarities and differences in the organisation of the sulfate assimilation pathway between basal and advanced land plants derived from genome comparisons of these two species with ARABIDOPSIS THALIANA and ORYZA SATIVA, two seed plants with sequenced genomes. We found differences in the number of genes encoding sulfate transporters, adenosine 5'-phosphosulfate reductase, and sulfite reductase between the lower and higher plants. The consequences for regulation of the pathway and evolution of sulfate assimilation in plants are discussed.

Sulfur metabolism in plants: are trees different?

Sulfur metabolite levels and sulfur metabolism have been studied in a significant number of herbaceous and woody plant species. However, only a limited number of datasets are comparable and can be used to identify similarities and differences between these two groups of plants. From these data, it appears that large differences in sulfur metabolite levels, as well as the genetic organization of sulfate assimilation and metabolism do not exist between herbaceous plants and trees. The general response of sulfur metabolism to internal and/or external stimuli, such as oxidative stress, seems to be conserved between the two groups of plants. Thus, it can be expected that, generally, the molecular mechanisms of regulation of sulfur metabolism will also be similar. However, significant differences have been found in fine tuning of the regulation of sulfur metabolism and in developmental regulation of sulfur metabolite levels. It seems that the homeostasis of sulfur metabolism in trees is more robust than in herbaceous plants and a greater change in conditions is necessary to initiate a response in trees. This view is consistent with the requirement for highly flexible defence strategies in woody plant species as a consequence of longevity. In addition, seasonal growth of perennial plants exerts changes in sulfur metabolite levels and regulation that currently are not understood. In this review, similarities and differences in sulfur metabolite levels, sulfur assimilation and its regulation are characterized and future areas of research are identified.

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

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.

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.

Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein.

Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria.

Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction.

The enzyme catalysing the reduction of adenosine 5'-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 degrees C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2-3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5'-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 degrees C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2-3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2-3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

Regulation of sulfate assimilation by nitrogen in Arabidopsis.

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5'-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO(3)(-), NH(4)(+), or glutamine (Gln), or 1 mM O-acetylserine (OAS). (35)SO(4)(2-) feeding showed that after addition of NH(4)(+), Gln, or OAS to nitrogen-starved plants, incorporation of (35)S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

Mutational analysis of 30 Slovenian cystic fibrosis patients compared to known Slovenian and European CF mutation spectra.

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of deltaF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT-->G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Adenosine 5'-phosphosulfate sulfotransferase and adenosine 5'-phosphosulfate reductase are identical enzymes.

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Identification, cloning, and properties of cytosolic D-ribulose-5-phosphate 3-epimerase from higher plants.

Plant cells contain a complete oxidative pentose phosphate pathway in the chloroplasts, but an incomplete pathway was proposed to be present in the cytosol, with cytosolic (cyt) isoforms of ribulose-5-phosphate 3-epimerase (RPEase) and other non-oxidative branch enzymes being undetectable. Here we present for the first time the identification, cloning, and properties of a cyt-RPEase in rice (Oryza sativa) and presence of its homologues in other plant species. Recombinant cyt-RPEase is a homodimer of 24.3-kDa subunits such as in the case of the animal and yeast enzymes, whereas the chloroplast (chl) RPEase is a hexamer. Cytosolic and chloroplastic RPEases cannot be separated by anion exchange chromatography. Since plant cyt-RPEase is more closely related in its primary structure to homologous enzymes in animal and yeast cells than to the chloroplast RPEase, the plant nuclear genes coding for cytosolic and chloroplast RPEases were most likely derived from eubacteria and cyanobacteria, respectively. Accumulation of cyt-RPEase-mRNA and protein is high in root cells, lacking chl-RPEase, and lower in green tissue. These and other observations support the view that green and non-green plant cells possess a complete oxidative pentose phosphate pathway in the cytosol.

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.

Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts.

Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization.

H-protein of the glycine cleavage system in Flaveria: alternative splicing of the pre-mRNA occurs exclusively in advanced C4 species of the genus.

A recent paper reports on the occurrence of alternative splicing of H-protein pre-mRNA in the C4 species, Flaveria trinervia, that is organ-specifically regulated. The analysis of 11 other species of the genus, F. cronquistii and F. pringlei (C3), F. anomala, F. chloraefolia, F. floridana, F. linearis and F. pubescens (C3-C4 intermediate), F. brownii (C4-like), F. palmeri, F. bidentis and F. australasica (C4), revealed that this post-transcriptional effect is not specific for F. trinervia. It occurs in all the examined C4 species of the genus Flaveria except the less advanced C4-like species, F. brownii. Both the position and the direct effect of alternative splicing, the addition of two alanine residues near to the N-terminus of the derived mature H-protein, are invariant. A quantification of the relative amounts of both transcripts revealed that, as in F. trinervia, the alternative mRNA strongly dominates in leaves. In sharp contrast, none of the C3, C3-C4 intermediate, or C4-like species showed alternative splicing. By Western analysis both H-isoproteins have been detected in F. trinervia and their ratio approximately corresponds to the measured transcript levels. It is concluded that alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system in all advanced Flaveria C4 species.

Chloroplast pentose-5-phosphate 3-epimerase from potato: cloning, cDNA sequence, and tissue-specific enzyme accumulation.

A cDNA clone encoding the chloroplast enzyme pentose-5-phosphate 3-epimerase (EC 5.1.3.1) in potato (Solanum tuberosum) was isolated and sequenced. The deduced sequence of 235 amino acids is similar to protein sequences of bacterial epimerases. Northern blot analysis showed the highest level of epimerase mRNA expression in potato leaves, whereas it was low in roots, tubers, and stems. Epimerase protein is mulated only in plant tissues possessing chloroplasts, i.e. in land to a lesser extent in stem. In contrast, transketolase, a sequential enzyme of epimerase in the reductive and oxidative pentose phosphate cycle, is accumulated in all plant tissues.

H-protein of glycine decarboxylase is encoded by multigene families in Flaveria pringlei and F. cronquistii (Asteraceae).

In Flaveria pringlei and F. cronquistii unlike other plants, H-protein of the glycine cleavage system is encoded by small multigene families. From leaf cDNA libraries and by reverse transcription of mRNA with subsequent polymerase chain reaction (PCR) amplification, we have obtained three different H-protein cDNA clones from each species. The relative levels of total H-protein mRNA, as well as of different H-protein transcripts, have been determined in leaves, stems, and roots of F. pringlei. Stems, with a total of 22% relative to leaves, contain substantial amounts of H-protein transcripts. The corresponding level in roots is relatively low (2.3% relative to leaves) but easily detectable. One of the transcripts occurs only in leaves (HFP20) and another one (HFP13) is present exclusively in photosynthesizing organs. Only one of the H-protein transcripts (HFP4) was found in all three organs, in leaves, stems, and roots of F. pringlei.

Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei.

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.

Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia.

Alternative splicing is a well-known post-transcriptional regulatory mechanism in eukaryotic organisms but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase multi-enzyme complex from the C4 species Flaveria trinervia revealed the presence of two transcript populations that differ in the length of their coding regions by six nucleotides. Otherwise, including their 3' nontranslated region, they are identical. From a genomic Southern analysis and from the sequencing of several independent cDNA clones it is evident that both types of transcript are derived from a single-copy gene. This gene, FTgdcsH, has been cloned and sequenced. It comprises four short exons. The two alternative splice sites are located at the end of intron 1. The shorter transcript closely corresponds to published H-protein mRNA sequences from other organisms. The longer transcript encodes two additional alanine residues very close to the N-terminus of the mature H-protein. A quantification of the relative amounts of both transcripts in different organs revealed that, with 80-90% of the total H-protein mRNA, the alternative mRNA dominates in leaves whereas roots contain more of the 'correctly' spliced transcript. It is concluded that, in F. trinervia and with a distinct organ preference, alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system.

T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase.

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.