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S'adenosyl-L: -methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene, polyamines, biotin, and nicotianamine in plants. Only limited information is available regarding its synthesis (SAM cycle) and its concentrations in plant tissues. The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight. Troughs of SAM levels were measured in the evening and night, which corresponds to the time when the major floral scent compound, methyl benzoate, is synthesized by a SAM dependent methyltransferase (NsBSMT) and when this enzyme possesses its highest activity. The SAM synthetase (NsSAMS1) and methionine synthase (NsMS1) are enzymes, among others, which are involved in the synthesis and regeneration of SAM. Respective genes were isolated from a N. suaveolens petal cDNA library. Transcript accumulation patterns of both SAM regenerating enzymes matched perfectly those of the bifunctional NsBSMT; maximum mRNA accumulations of NsMS1 and NsSAMS1 were attained in the evening. Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers. It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E(GSH)) in the cytosol. Using this tool, it is shown that E(GSH) in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.

Nicotianamine chelates and transports micronutrient metal ions in plants. It has been speculated that nicotianamine is involved in seed loading with micronutrients. A tomato (Solanum lycopersicum) mutant (chloronerva) and a tobacco (Nicotiana tabacum) transgenic line have been utilized to analyze the effects of nicotianamine loss. These mutants showed early leaf chlorosis and had sterile flowers. Arabidopsis (Arabidopsis thaliana) has four NICOTIANAMINE SYNTHASE (NAS) genes. We constructed two quadruple nas mutants: one had full loss of NAS function, was sterile, and showed a chloronerva-like phenotype (nas4x-2); another mutant, with intermediate phenotype (nas4x-1), developed chlorotic leaves, which became severe upon transition from the vegetative to the reproductive phase and upon iron (Fe) deficiency. Residual nicotianamine levels were sufficient to sustain the life cycle. Therefore, the nas4x-1 mutant enabled us to study late nicotianamine functions. This mutant had no detectable nicotianamine in rosette leaves of the reproductive stage but low nicotianamine levels in vegetative rosette leaves and seeds. Fe accumulated in the rosette leaves, while less Fe was present in flowers and seeds. Leaves, roots, and flowers showed symptoms of Fe deficiency, whereas leaves also showed signs of sufficient Fe supply, as revealed by molecular-physiological analysis. The mutant was not able to fully mobilize Fe to sustain Fe supply of flowers and seeds in the normal way. Thus, nicotianamine is needed for correct supply of seeds with Fe. These results are fundamental for plant manipulation approaches to modify Fe homeostasis regulation through alterations of NAS genes.

The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5'-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3'-phosphoadenosine 5'-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation.

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for AtERD2 with six transmembrane domains.

Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.

The effects of chromate on sulfate uptake and assimilation were investigated in the accumulator Brassica juncea (L.) Czern. Seven-day-old plants were grown for 2 d under the following combination of sulfate and chromate concentration: (i) no sulfate and no chromate (-S), (ii) no sulfate and 0.2 mmol L(-1) chromate (-S +Cr), (iii) 1 mmol L(-1) sulfate and no chromate (+S), or (iv) 1 mmol L(-1) sulfate and 0.2 mmol L(-1) chromate (+S +Cr). Despite the toxic effects exerted by chromate as indicated by altered level of reducing sugars and proteins in leaves, the growth of B. juncea was only weakly reduced by chromate, and no variation in chlorophyll a and b was measured, regardless of S availability. Chromium (Cr) was stored more in roots than in leaves, and the maximum Cr accumulation was measured in -S +Cr plants. The significant decrease of the sulfate uptake rates observed in Cr-treated plants was accompanied by a repression of the root low-affinity sulfate transporter (BjST1), suggesting that the transport of chromate in B. juncea may involve sulfate carriers. Once absorbed, chromate induced genes involved in sulfate assimilation (ATP-sulfurylase: atps6; APS-reductase: apsr2; Glutathione synthethase: gsh2) and accumulation of cysteine and glutathione, which may suggest that these reduced S compounds play a role in Cr tolerance. Together, our findings indicate that when phytoremediation technologies are used to recover Cr-contaminated areas, the concentration of sulfate in the plant growth medium must be considered because it may influence the ability of plants to accumulate and tolerate Cr.

The molecular mechanisms regulating the initial uptake of inorganic sulfate in plants are still largely unknown. The current model for the regulation of sulfate uptake and assimilation attributes positive and negative regulatory roles to O-acetyl-serine (O-acetyl-Ser) and glutathione, respectively. This model seems to suffer from exceptions and it has not yet been clearly validated whether intracellular O-acetyl-Ser and glutathione levels have impacts on regulation. The transcript level of the two high-affinity sulfate transporters SULTR1.1 and SULTR1.2 responsible for sulfate uptake from the soil solution was compared to the intracellular contents of O-acetyl-Ser, glutathione, and sulfate in roots of plants submitted to a wide diversity of experimental conditions. SULTR1.1 and SULTR1.2 were differentially expressed and neither of the genes was regulated in accordance with the current model. The SULTR1.1 transcript level was mainly altered in response to the sulfur-related treatments. Split-root experiments show that the expression of SULTR1.1 is locally regulated in response to sulfate starvation. In contrast, accumulation of SULTR1.2 transcripts appeared to be mainly related to metabolic demand and is controlled by photoperiod. On the basis of the new molecular insights provided in this study, we suggest that the expression of the two transporters depends on different regulatory networks. We hypothesize that interplay between SULTR1.1 and SULTR1.2 transporters could be an important mechanism to regulate sulfate content in the roots.

Background: The shape of the dose-response curve at low doses differs from the linear quadratic model. The effect of a radio-adaptive response is the centre of many studies and well known inspite that the clinical applications are still rarely considered., Methods: We studied the effect of a low-dose pre-irradiation (0.03 Gy - 0.1 Gy) alone or followed by a 2.0 Gy challenging dose 4 h later on the survival of the HT29 cell line (human colorectal cancer cells) and on the GM637 cell line (human fibroblasts)., Results: 0.03 Gy given alone did not have a significant effect on both cell lines, the other low doses alone significantly reduced the cell survival. Applied 4 h before the 2.0 Gy fraction, 0.03 Gy led to a significant induced radioresistance in GM637 cells, but not in HT29 cells, and 0.05 Gy led to a significant hyperradiosensitivity in HT29 cells, but not in GM637 cells., Conclusion: A pre-irradiation with 0.03 Gy can protect normal fibroblasts, but not colorectal cancer cells, from damage induced by an irradiation of 2.0 Gy and the application of 0.05 Gy prior to the 2.0 Gy fraction can enhance the cell killing of colorectal cancer cells while not additionally damaging normal fibroblasts. If these findings prove to be true in vivo as well this may optimize the balance between local tumour control and injury to normal tissue in modern radiotherapy.

Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, gamma-glutamate cysteine ligase (GSH1), responsible for synthesis of gamma-glutamylcysteine (gamma-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by gamma-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of gamma-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by gamma-EC in vitro strongly suggests export of gamma-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, gamma-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL-like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.

The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.

Sulphur dioxide (SO(2)) is known as a strongly damaging air pollutant. After conversion to sulphite in aqueous solution, it becomes a strong nucleophilic agent that attacks numerous compounds in the cell. Therefore, plants have developed a mechanism to control sulphite levels. Recently, we have cloned and characterized the enzyme sulphite oxidase (SO) from Arabidopsis thaliana. Yet, its physiological role remained unclear. Here, we describe results demonstrating that SO is essential for detoxifying excessive amounts of sulphite in the cell which is important for the survival of the plant. T-DNA-tagged A. thaliana plants lacking the enzyme showed a decrease in vitality during SO(2) fumigation and a change in their S-metabolites. The same was found with RNA-interference (RNAi) plants that were generated for tobacco. On the contrary, over-expression of SO helped the plant to survive SO(2) concentrations that are detrimental for non-transformed wild-type (WT) plants, as was shown with poplar plants which are known to be particularly sensitive to SO(2). Fumigation induced the expression of the enzyme as demonstrated by promoter-reporter gene fusion, by immunoblot analysis of SO-protein and by induction of enzyme activity. This implies that SO, as an otherwise constitutively expressed protein, is under additional control by SO(2) in the environment.

Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.

The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione-bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the gamma-glutamylcysteine-bimane intermediate (gamma-EC-B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine-bimane without intermediate. Only co-infiltration of monochlorobimane with Cd2+ and Cu2+ increased gamma-EC-B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null-mutants. In the absence of elevated heavy metal concentrations glutathione-conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate-limiting.

Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric cysteine synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.

Sulfur is one of the critical elements in living matter, as it participates in several structural, metabolic and catalytic activities. Photosynthesis is an important process that entails the use of sulfur during both the light and carbon reactions. Nearly half of global photosynthetic carbon fixation is carried out by phytoplankton in the aquatic environment. Aquatic environments are very different from one another with respect to sulfur content: while in the oceans sulfate concentration is constantly high, freshwaters are characterized by daily and seasonal variations and by a wide range of sulfur concentration. The strategies that algal cells adopt for energy and resource allocation often reflect these differences. In the oceans, the amount and chemical form of sulfur has changed substantially during the course of the Earth's history; it is possible that sulfur availability played a role in the evolution of marine phytoplankton communities and it may continue to have appreciable effects on global biogeochemistry and ecology. Phytoplankton is also the main biogenic source of sulfur; sulfur can be released into the atmosphere by algal cells as dimethylsulfide, with possibly important repercussions on global climate. These and related matters are discussed in this review.

Continuous control of metabolism and developmental processes is a key feature of live cells. Cysteine thiol residues of proteins are both exceptionally useful in terms of structural and regulatory aspects, but at the same time exceptionally vulnerable to oxidation. Conserved cysteines thus are highly important for the function of metabolic enzymes and for signaling processes underlying responses to environmental factors. The underlying mechanism for the central role of thiol-mediated redox control in cellular metabolism is the ability of the cysteine-thiols to reversibly change their redox state followed by changes of structural, catalytic or regulatory functions. The cellular glutathione/glutathione disulfide redox buffer is present in cells at millimolar concentrations and forms one major basis of redox homeostasis by which protein thiols can maintain their redox state or oxidized protein thiols can be reverted to their reduced state. Besides acting as redox buffer, glutathione also acts as an electron donor for both scavenging of reactive oxygen, e.g. from photosynthesis and respiration, and metabolic reactions such as reduction of hydroperoxides and lipidperoxides or sulfate assimilation. The central role of glutathione is further emphasized by its involvement in signaling processes and the crosstalk of redox signaling processes with other means of signaling including protein glutathionylation and control of transcription factors. The present review aims at highlighting the key functions of glutathione in thiol-mediated redox control and its interplay with other protein-thiol-based redox systems.

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2-fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.

During ontogeny of barley plants (Hordeum vulgare, L. cv. Barke), a continuous developmental gradient of new leaves at the top and lower leaves undergoing senescence is maintained. In the course of senescence, specific recycling processes efficiently transfer valuable resources, e.g. nitrogen and carbon, to the growing young leaves and ears. In order to understand the temporal and spatial sequence of processes underlying this developmental program of leaf formation and degradation, changes in photosynthetic parameters, as well as C and N levels of all individual leaves were determined. During whole plant ontogeny, a strict sequential pattern of incorporation and degradation of C and N resources in the individual leaves, accompanied by a sequential loss of chlorophyll and photosynthetic function, was observed. In addition, protein levels of key enzymes of C and N anabolism AGPase (ADPglucose pyrophosphorylase) and GS (glutamine synthetase; plastidic isoform) also showed a strict pattern of sequential down-regulation in senescing leaves. Their decline preceded the breakdown of chlorophyll, total C and N levels and photosynthetic performance in the leaves. Quantitative real time PCR measurements revealed that the down-regulation of protein content of AGPase and GS correlated with a drastic decrease in their transcript levels. These data elucidated precise temporal and spatial regulation of C and N metabolism and allocation with photosynthetic function in the leaves during whole plant ontogeny of barley.

Sulfur emission from marine phytoplankton has been recognized as an important factor for global climate and as an entry into the biogeochemical S cycle. Despite this significance, little is known about the cellular S metabolism in algae that forms the basis of this emission. Some biochemical and genetic evidence for regulation of S uptake and assimilation is available for the freshwater model alga Chlamydomonas. However, the marine environment is substantially different from most fresh waters, containing up to 50 times higher free sulfate concentrations and challenging the adaptive mechanisms of primary and secondary S metabolism in marine algae. This review intends to integrate ecological and physiological data to provide a comprehensive view of the role of S in the oceans.

Excitation imbalances between photosystem I and II generate redox signals in the thylakoid membrane of higher plants which induce acclimatory changes in the structure of the photosynthetic apparatus. They affect the accumulation of reaction center and light-harvesting proteins as well as chlorophylls a and b. In Arabidopsis thaliana the re-adjustment of photosystem stoichiometry is mainly mediated by changes in the number of photosystem I complexes, which are accompanied by corresponding changes in transcripts for plastid reaction center genes. Because chloroplast protein complexes contain also many nuclear encoded components we analyzed the impact of such photosynthetic redox signals on nuclear genes. Light shift experiments combined with application of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea have been performed to induce defined redox signals in the thylakoid membrane. Using DNA macroarrays we assessed the impact of such redox signals on the expression of nuclear genes for chloroplast proteins. In addition, studies on mutants with lesions in cytosolic photoreceptors or in chloroplast-to-nucleus communication indicate that the defective components in the mutants are not essential for the perception and/or transduction of light-induced redox signals. A stable redox state of glutathione suggest that neither glutathione itself nor reactive oxygen species are involved in the observed regulation events pointing to the thylakoid membrane as the main origin of the regulatory pathways. Our data indicate a distinct role of photosynthetic redox signals in the cellular network regulating plant gene expression. These redox signals appear to act independently and/or above of cytosolic photoreceptor or known chloroplast-to-nucleus communication avenues.

Ser acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetyl-Ser from L-Ser and acetyl-CoA, leading to synthesis of Cys. According to its position at the decisive junction of the pathways of sulfur assimilation and amino acid metabolism, SATases are subject to regulatory mechanisms to control the flux of Cys synthesis. In Arabidopsis (Arabidopsis thaliana) there are five genes encoding SATase-like proteins. Two isoforms, Serat3;1 and Serat3;2, were characterized with respect to their enzymatic properties, feedback inhibition by L-Cys, and subcellular localization. Functional identity of Serat3;1 and Serat3;2 was established by complementation of a SATase-deficient mutant of Escherichia coli. Cytosolic localization of Serat3;1 and Serat3;2 was confirmed by using fusion construct with the green fluorescent protein. Recombinant Serat3;1 was not inhibited by L-Cys, while Serat3;2 was a strongly feedback-inhibited isoform. Quantification of expression patterns indicated that Serat2;1 is the dominant form expressed in most tissues examined, followed by Serat1;1 and Serat2;2. Although Serat3;1 and Serat3;2 were expressed weakly in most tissues, Serat3;2 expression was significantly induced under sulfur deficiency and cadmium stress as well as during generative developmental stages, implying that Serat3;1 and Serat3;2 have specific roles when plants are subjected to distinct conditions. Transgenic Arabidopsis plants expressing the green fluorescent protein under the control of the five promoters indicated that, in all Serat genes, the expression was predominantly localized in the vascular system, notably in the phloem. These results demonstrate that Arabidopsis employs a complex array of compartment-specific SATase isoforms with distinct enzymatic properties and expression patterns to ensure the provision of Cys in response to developmental and environmental changes.

Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.

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.

The synthesis of cysteine is positioned at a decisive stage of assimilatory sulphate reduction, marking the fixation of inorganic sulphide into a carbon skeleton. O-acetylserine (thiol) lyase (OAS-TL) catalyses the reaction of inorganic sulphide with O-acetylserine (OAS). Despite its prominent position in the pathway OAS-TL is generally regarded as a non-limiting enzyme without regulatory function, due to low substrate affinities and semi-constitutive expression patterns. To resolve this apparent contradiction, the kinetic properties of three OAS-TLs from Arabidopsis thaliana, localized in the cytosol (A), plastids (B), and mitochondria (C), were analysed. The recombinant expressed OAS-TLs were purified to apparent homogeneity without any fusion tag to maintain their native forms. The proteins displayed high specific activities of 550-900 micromol min(-1) mg(-1). Using an improved and highly sensitive assay method for cysteine determination, the apparent K(m)(sulphide) was 3-6 microM for OAS-TL A, B, and C and thus 10-100 times lower than previously reported for plant OAS-TLs. K(m)(OAS) was between 310 microM and 690 microM for OAS-TL isoform A, B, and C, whereas the apparent dissociation binding constant for OAS was much lower (K(d)<1 microM OAS). A HPLC method was developed for OAS quantification that revealed fast increases of the cellular OAS concentration in response to sulphate deprivation. The observed fluctuations of intracellular OAS concentrations, combined with the OAS dissociation constant and the catalytic properties of OAS-TL, support the model of a dynamic cysteine synthesis system with regulatory function as can be expected from the position of the reaction in the sulphur assimilation pathway.

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.

Iron is an essential micronutrient with numerous cellular functions, and its deficiency represents one of the most serious problems in human nutrition worldwide. Plants have two major problems with iron as a free ion: its insolubility and its toxicity. To ensure iron acquisition from soil and to avoid iron excess in the cells, uptake and homeostasis are tightly controlled. Plants meet the extreme insolubility of oxidized iron at neutral pH values by deficiency-inducible chelation and reduction systems at the root surface that facilitate uptake. Inside the cells the generation of highly toxic hydroxyl radicals by iron redox changes is avoided by intricate chelation mechanisms. Organic acids, most notably nicotianamine, and specialized proteins bind iron before it can be inserted into target molecules for biological function. Uptake and trafficking of iron throughout the plant is therefore a highly integrated process of membrane transport and reduction, trafficking between chelator species, whole-plant allocation and genetic regulation. The improvement of crop plants with respect to iron efficiency on iron-limiting soils and to iron fortification for human nutrition has been initiated by breeding and biotechnology. These efforts have to consider molecular and physiological evidence to overcome the inherent barriers and problems of iron metabolism.

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

Tobacco (Nicotiana tabacum L.) plants were subjected to a prolonged period of sulfur-deprivation to characterize molecular and metabolic mechanisms that permit control of primary N-metabolism under these conditions. Prior to the appearance of chlorotic lesions, sulfur-deprived tobacco leaves showed a strong decrease in the sulfate content and changes in foliar enzyme activities, mRNA accumulation and amino-acid pools. The basic amino acids glutamine, asparagine and arginine accumulated in the leaves of sulfur-deprived plants, while the foliar concentrations of aspartate, glutamate, serine or alanine remained fairly unchanged. Maximal extractable nitrate reductase (NR; EC 1.6.6.1) activity decreased strongly in response to sulfur-deprivation. The decrease in maximal extractable NR activity was accompanied by a decline in NR transcripts while the mRNAs of the plastidic glutamine synthetase (EC 6.1.3.2) or the beta-subunit of the mitochondrial ATP synthase were much less affected. Nitrate first accumulated in leaves of tobacco during sulfur-deprivation but then declined. An appreciable amount of nitrate was, however, present in severely sulfur-depleted leaves. The repression of NR gene expression is, therefore, not related to the decrease in the leaf nitrate level. However, glutamine- and/or asparagine-mediated repression of NR gene transcription is a possible mechanism of control in situations when glutamine and asparagine accumulate in leaves and provides a feasible explanation for the reduction in NR activity during sulfur-deprivation. The removal of reduced nitrogen from primary metabolism by redirection and storage as arginine, asparagine or glutamine combined with the down-regulation of nitrate reduction via glutamine- and/or asparagine-mediated repression of NR gene transcription may contribute to maintaining a normal N/S balance during sulfur-deprivation and indicate that the co-ordination of N- and S-metabolism is retained under these conditions.

The biosynthesis of cysteine represents the final step of sulfate assimilation in bacteria and plants. It is catalyzed by the sequential action of serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which form a cysteine synthase (CS) complex in vitro. SAT and OAS-TL from Arabidopsis thaliana have previously been cloned, and now the first evidence is presented for the CS complex and SAT self-interaction in vivo employing the yeast two-hybrid system. Application of this method proved to be an efficient tool for the analysis of protein-protein interactions within a plant metabolic protein complex. Mapping of SAT domain structure revealed two new, independent domains with specific functions in protein-protein interaction. Analysis using truncated proteins proved the C-terminus of SAT to be sufficient for association with OAS-TL and to correlate with the putative transferase activity domain. SAT/SAT interaction was localized in the central region of the protein and occurred also between SAT isoforms. Both protein interaction domains coincided with distinct alpha-helical and beta-sheet clusters and together correlated with the minimal protein structure required for SAT catalysis as shown by functional complementation of an Escherichia coli mutant. The homo- and hetero-oligomerization properties are discussed with respect to the assumed function of the CS complex in metabolic channeling and activation of SAT by interaction with OAS-TL.

The regulation by photorespiration of the transcript level corresponding to plastidic glutamine synthetase (GS-2) was investigated in the leaves of Arabidopsis thaliana (L.) Heynh.. Photorespiration was suppressed by growing the plants in an atmosphere containing 300 Pa CO2. Suppression of photorespiration was demonstrated by the ability of the conditionally lethal serine hydroxymethyltransferase (SHMT)-deficient stm mutant of A. thaliana to grown normally under these conditions. In contrast to previous studies with bean or pea that were performed at very high CO2 partial pressure (2-4 kPa; Edwards and Coruzzi, 1989, Plant Cell 1:241-248; Cock et al., 1991, Plant Mol Biol 17: 761-771), suppression of photorespiration during growth of A. thaliana in an atmosphere with 300 Pa CO2 had no effect of the leaf GS-2 transcript level. In the short term, neither suppression of photorespiration induced by the transfer of air-grown A. thaliana plants into a CO2-enriched atmosphere, nor an increase in the rate of photorespiration achieved by the transfer of high-CO2-grown A. thaliana plants into air resulted in a change in the GS-2 mRNA level. The absence of photorespiratory ammonium release in leaves of the stm mutant had no effect on the GS-2 transcript level. Overall, our data argue against a control by photorespiration of the A. thaliana leaf GS-2 mRNA pool. In contrast, regulation of the leaf SHMT mRNA level may involve a negative feedback effect of at least one metabolite derived from the glycine/serine conversion during photorespiration, as indicated by the overexpression of SHMT transcripts in the leaves of the stm mutant.

A cDNA encoding for serine acetyltransferase which catalyzes the committing step of cysteine biosynthesis has been cloned from Arabidopsis thaliana. The plant protein has a predicted molecular weight of 32.8 kDa and shows up to 43% of amino acid homology to bacterial serine acetyltransferases. It complements a serine acetyltransferase negative E. coli mutant and can be enzymatically determined in the heterologous host. The corresponding mRNA is predominantly expressed in light exposed tissue and represents one of at least two related genes.

cDNAs encoding for two isoforms of O-acetylserine (thiol) lyase (OAS-TL), which catalyzes the synthesis of cysteine, have been isolated from Arabidopsis thaliana. Secondary structure together with expression patterns derived during photomorphogenesis indicate cellular localizations in the cytosol and plastids, thus allowing a direct comparison of compartment-specific forms within one species. The cytosolic OAS-TL complemented an E. coli auxotrophic mutant lacking cysteine synthesis. Both isoforms are represented by small gene families. They are expressed under all conditions investigated and were observed to increase in expression in plants grown with limited sulfate supply.

λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg(2+). With 0.2mM Cd(2+) or Zn(2+), enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andL-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent Km values forL-glutamate,L-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell.

Human acrosin was purified to electrophoretically homogeneous forms by acidic extraction of washed ejaculated spermatozoa and gel filtration of the acidic extracts on Sephadex G-75, followed by affinity chromatography on p-amino-benzamidine Sepharose. Human acrosin exists in at least four molecular forms. The apparent molecular weights of three forms were determined to be 64 000, 38 000 and 25 000, respectively. The high molecular weight form is transformed to the low molecular weight forms by incubation of the acrosin preparation obtained from freshly ejaculated spermatozoa in solutions of pH near 7. Like boar acrosin, human acrosin is also a glycoprotein and therefore reversibly bound to Concanavalin A-Sepharose. The amino acid composition of the 25 000 molecular weight form is similar to that of human trypsin. Rabbit anti-boar-acrosin gamma-globulins form a precipitate with human acrosin, but not with porcine trypsin or human plasmin. The relationship between the occurrence of multiple acrosin forms and proenzyme activation by limited proteolysis is discussed.

A high performance liquid chromatography system was developed for the quantitative separation and determination of bradykinin, kallidin and Met-Lys-bradykinin using a reversed-phase column (Nucleosil 5 C8) and an isocratic buffer system. The lower limit of detection is 20 pmol for bradykinin. The intra-variation coefficient of the assay is between 1.8 and 5.7%. The recovery of bradykinin is higher than 96%. Addition of human serum albumin to the assay system in a concentration of 40% lead to a remarkable reduction of the detectable kinin level, which indicates an adsorption of kinins to serum albumin.

Boar acrosin was isolated to electrophoretic homogeneity by modification of the method described by us previously. Titration with p-nitrophenyl p-guanidinobenzoate revealed a molar activity of 8900 toward BzArgOEt and of 780 towards BzArgNHNp. Treatment with neuraminidase led to an altered electrophoretic mobility, indicating that acrosin is sialogycoprotein

Further evidence is presented that the acrosomal proteinase acrosin exists as a zymogen precursor in freshly ejaculated boar spermatozoa. Autoactivation of proacrosin to acrosin takes place optimally at slightly alkaline pH and in the presence of calcium ions. Activation is considerably accelerated by catalytic amounts of trypsin or highly purified acrosin. A significant acceleration of the activation is also achieved by porcine pancreatic and urinary kallikrein, whereas chymotrypsin, plasmin, thrombin or urokinase showed no effect. Activation can be inhibited by p-amino-benzamidine and p-nitrophenyl p'-guanidino-benzoate. Electrophoretic analysis at different stages of activation revealed that during this process various molecular forms of acrosin are produced, apparently by limited proteolysis.

The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis.