Ruprecht-Karls-Universität Heidelberg
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Molekulare Biologie der Pflanzen

Dr. Markus Wirtz

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Publications in Peer-reviewed journals

| 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2005 | 2004 | 2003 | 2002 | 2001 | 2000

 

2015

Linster, E., Stephan, I., Bienvenut, W.V., Maple-Grodem, J., Myklebust, L.M.., Huber, M., Reichelt, M., Sticht, C., Geir Moller, S., Meinnel, T., Arnesen, T., Giglione, C., Hell, R., Wirtz, M. (2015).
Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis.
Nature Commun, 6: 7640.
Abstract
N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid.
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Xu, F., Huang, Y., Li, L., Gannon, P., Linster, E., Huber, M., Kapos, P., Bienvenut, W., Polevoda, B., Meinnel, T., Hell, R., Giglione, C., Zhang, Y., Wirtz, M., Chen, S., Li, X. (2015).
Two N-terminal acetyltransferases antagonistically regulate the stability of a nod-like receptor in Arabidopsis
Plant Cell, 27: 1547-62.
Abstract
Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.
Abstract 
Timm, S., Wittmiss, M., Gamlien, S., Ewald, R., Florian, A., Frank, M., Wirtz, M., Hell, R., Fernie, A. R., Bauwe, H. (2015).
Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.
Plant Cell, epub.
Abstract
Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain alpha-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)+-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency.
Abstract 
Dinh, T. V., Bienvenut, W. V., Linster, E., Feldman-Salit, A., Jung, V. A., Meinnel, T. Hell, R., Giglione, C., Wirtz, M. (2015).
Molecular identification and functional characterization of the first N-alpha-acetyltransferase in plastids by global acetylome profiling.
Proteomics, 15: 2426-2435.
Abstract
Protein N-alpha-terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N-alpha-acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein Nalpha -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays Nepsilon -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947).
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Yang. Y., M Pollard, A., Höfler, C., Poschet, G., Wirtz, M., Hell, R., Sourjik, V. (2015).
Relation between chemotaxis and consumption of amino acids in bacteria.
Mol Microbiol., 96: 1272-1282.
Abstract
Chemotaxis enables bacteria to navigate chemical gradients in their environment, accumulating toward high concentrations of attractants and avoiding high concentrations of repellents. Although finding nutrients is likely to be an important function of bacterial chemotaxis, not all characterized attractants are nutrients. Moreover, even for potential nutrients, the exact relation between the metabolic value of chemicals and their efficiency as chemoattractants has not been systematically explored. Here we compare the chemotactic response of amino acids with their use by bacteria for two well-established models of chemotactic behavior, Escherichia coli and Bacillus subtilis. We demonstrate that in E.?coli chemotaxis toward amino acids indeed strongly correlates with their utilization. However, no such correlation is observed for B.?subtilis, suggesting that in this case, the amino acids are not followed because of their nutritional value but rather as environmental cues.
PubMed 
Birke, H., Hildebrandt, T. M., Wirtz, M., Hell, R. (2015).
Sulfide detoxification in plant mitochondria.
Methods Enzymol, 555: 271-286.
Abstract
In contrast to animals, which release the signal molecule sulfide in small amounts from cysteine and its derivates, phototrophic eukaryotes generate sulfide as an essential intermediate of the sulfur assimilation pathway. Additionally, iron-sulfur cluster turnover and cyanide detoxification might contribute to the release of sulfide in mitochondria. However, sulfide is a potent inhibitor of cytochrome c oxidase in mitochondria. Thus, efficient sulfide detoxification mechanisms are required in mitochondria to ensure adequate energy production and consequently survival of the plant cell. Two enzymes have been recently described to catalyze sulfide detoxification in mitochondria of Arabidopsis thaliana, O-acetylserine(thiol)lyase C (OAS-TL C), and the sulfur dioxygenase (SDO) ethylmalonic encephalopathy protein 1 (ETHE1). Biochemical characterization of sulfide producing and consuming enzymes in mitochondria of plants is fundamental to understand the regulatory network that enables mitochondrial sulfide homeostasis under nonstressed and stressed conditions. In this chapter, we provide established protocols to determine the activity of the sulfide releasing enzyme beta-cyanoalanine synthase as well as sulfide-consuming enzymes OAS-TL and SDO. Additionally, we describe a reliable and efficient method to purify OAS-TL proteins from plant material.
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Tavares, S., Wirtz, M., Beier, M. P., Bogs, J., et al. (2015).
Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants.
Front Plant Sci 2015, 6: 74.
Abstract
In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.
Speiser, A., Haberland, S., Watanabe, M., Wirtz, M., et al. (2015).
The significance of cysteine synthesis for acclimation to high light conditions.
Front Plant Sci 2014, 5, 776.
Abstract
Situations of excess light intensity are known to result in the emergence of reactive oxygen species that originate from the electron transport chain in chloroplasts. The redox state of glutathione and its biosynthesis contribute importantly to the plant's response to this stress. In this study we analyzed the significance of cysteine synthesis for long-term acclimation to high light conditions in Arabidopsis thaliana. Emphasis was put on the rate-limiting step of cysteine synthesis, the formation of the precursor O-acetylserine (OAS) that is catalyzed by serine acetyltransferase (SERAT). Wild type Arabidopsis plants responded to the high light condition (800 mumol m(-2) s(-1) for 10 days) with synthesis of photo-protective anthocyanins, induction of total SERAT activity and elevated glutathione levels when compared to the control condition (100 mumol m(-2) s(-1)). The role of cysteine synthesis in chloroplasts was probed in mutant plants lacking the chloroplast isoform SERAT2;1 (serat2;1) and two knock-out alleles of CYP20-3, a positive interactor of SERAT in the chloroplast. Acclimation to high light resulted in a smaller growth enhancement than wild type in the serat2;1 and cyp20-3 mutants, less induction of total SERAT activity and OAS levels but similar cysteine and glutathione concentrations. Expression analysis revealed no increase in mRNA of the chloroplast SERAT2;1 encoding SERAT2;1 gene but up to 4.4-fold elevated SERAT2;2 mRNA levels for the mitochondrial SERAT isoform. Thus, lack of chloroplast SERAT2;1 activity or its activation by CYP20-3 prevents the full growth response to high light conditions, but the enhanced demand for glutathione is likely mediated by synthesis of OAS in the mitochondria. In conclusion, cysteine synthesis in the chloroplast is important for performance but is dispensable for survival under long-term exposure to high light and can be partially complemented by cysteine synthesis in mitochondria.
Birke H, De Kok LJ, Wirtz M, Hell R. (2015).
The Role of Compartment-Specific Cysteine Synthesis for Sulfur Homeostasis During H2S Exposure in Arabidopsis.
Plant Cell Physiol. 65: 358-367. 
Abstract
Sulfide is the end-product of assimilatory sulfate reduction in chloroplasts. It is then used by O-acetylserine(thiol)lyase (OAS-TL) to produce cysteine, the source of reduced sulfur in plants. While its formation in chloroplasts is essential for plant metabolism, sulfide is also a potent toxin mainly targeting respiration in mitochondria. Here, the application of sublethal concentrations of sulfide to Arabidopsis thaliana was used to by-pass assimilatory sulfate reduction, resulting in down-regulation of most genes of the pathway. The dualism of sulfide as substrate and toxin was investigated using knock-out mutants of the chloroplast-, mitochondrion- and cytosol-targeted OAS-TL isoforms. Surprisingly, growth retardation due to intoxication by sulfide was independent of the presence or absence of the three OAS-TL isoforms, indicating rapid exchange towards sulfur homoeostasis between the compartments. Cysteine, glutathione and sulfate, and less so S-sulfocysteine, were identified as major sinks for excess sulfide in wild-type plants. Additionally, the concentration of thiosulfate increased 1,000-fold, pointing towards a significant function of thiosulfate formation during H2S exposure. Synthesis of cysteine in the cytosol was found to be particularly important for accumulation of sulfite, sulfate and thiosulfate, indicating an important role for cytosolic OAS-TL for the re-oxidation of sulfide. The results show that thiosulfate and sulfate accumulation is strongly linked to cytosolic cysteine synthesis and that scavenging of sulfide by cysteine synthesis enhances sulfur compound accumulation. However, lack of cysteine synthesis in a subcellular compartment has no crucial consequences for toxicity and subsequent growth retardation.
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2014

Salbitani G, Wirtz M, Hell R., Carfagna, S (2014).
Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana.
Metabolites. 4: 629-639. 
Abstract
In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32–34 kDa and are thus slightly larger compared to those found in other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species.tween Fe and S nutritional status is evaluated. Experiments with Arabidopsis thaliana and crop plants indicate a co-regulation and point to a possible role of Fe-S cluster synthesis or abundance in the Fe/S network.
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Krüssel L, Junemann J, Wirtz M, Birke H, Thornton JD, Browning LW, Poschet G, Hell R, Balk J, Braun HP, Hildebrandt TM (2014).
The Mitochondrial Sulfur Dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 Is Required for Amino Acid Catabolism during Carbohydrate Starvation and Embryo Development in Arabidopsis.
Plant Physiol. 165: 92-104. 
Abstract
The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine.
Pubmed 
Cao M, Wang Z, Zhao Q, Mao J, Speiser A, Wirtz M, Hell R, Zhu J-K, Xiang CB. (2014).
Sulfate availability affects ABA levels and germination response to ABA and salt stress in Arabidopsis thaliana.
Plant J. 77: 604-615. 
Abstract
Sulfur containing compounds play a critical role in the response of plants to abiotic stress factors including drought. The phytohormone abscisic acid (ABA) is the key regulator of responses to drought and high salt stress. However, our knowledge about interaction of S-metabolism and ABA biosynthesis is scarce. Here we report that sulfate supply affects synthesis and steady-state levels of ABA in Arabidopsis wildtype seedlings. By using different mutants of the sulfate uptake and reduction pathway, we confirmed the impact of sulfate supply on steady-state ABA content in Arabidopsis and demonstrated that this impact was due to cysteine availability. Loss of the chloroplast sulfate transporter3;1 function (sultr3;1) resulted in significantly decreased aldehyde oxidase (AO) activity and ABA levels in seedlings and seeds. These mutant phenotypes could be reverted by exogenous application of cysteine or ectopic expression of SULTR3;1. In addition the sultr3;1 mutant showed a decrease of xanthine dehydrogenase activity, but not of nitrate reductase, strongly indicating that in seedlings cysteine availability limits activity of the molybdenum co-factor sulfurase, ABA3, which requires cysteine as the S-donor for sulfuration. Transcription of ABA3 and NCED3, encoding another key-enzyme of the ABA biosynthesis pathway, was regulated by S-supply in wildtype seedlings. Vice versa, ABA up-regulated the transcript level of SULTR3;1 and other S-metabolism related genes. Our results provide evidence for a significant co-regulation of S-metabolism and ABA biosynthesis that operates to ensure sufficient cysteine for AO maturation and highlight the importance of sulfur for stress tolerance of plants.
Pubmed 
Lee CP, Wirtz M, Hell R. (2014).
Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.
Plant Cell Physiol. 55: 64-73. 
Abstract
Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that externally-fed [35S]-cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organellar protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ?pH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.
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2013

Birke H, Wirtz M, Hell R. (2013).
Successful fertilization requires the presence of at least one major O-acetylserine(thiol)lyase for cysteine synthesis in pollen of Arabidopsis.
Plant Physiol. 163: 959-972. 
Abstract
Synthesis of cysteine is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis thaliana nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms OAS-TL A, OAS-TL B and OAS-TL C catalyze the formation of cysteine by combining O-acetylserine and sulfide in the cytosol, the plastids and the mitochondria, respectively. So far the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-offunction mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient cysteine synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses evidenced exclusiveness of cysteine synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demontrated that the presence of at least one functional OAS-TL isoform is essential for proper function of the male gametophyte, although synthesis of histidine, lysine and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed to separate distinct functions of cysteine and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of cysteine into the developing ovule from the mother plant.
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Cao MJ, Wang Z, Wirtz M, Hell R, Oliver DJ, Xiang CB. (2013).
SULTR3;1 is a chloroplast-localized sulfate transporter in Arabidopsis thaliana.
Plant J. 73: 607 - 616. 
Abstract
Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid-localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1-GFP localization, western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss-of-SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S-SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.
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Forieri I, Wirtz M, Hell R. (2013).
Towards new perspectives on the interaction of iron and sulfur metabolism in plants.
Front Plant Sci. Oct 2;4:357. doi: 10.3389/fpls.2013.00357. 
Abstract
The deficiency of nutrients has been extensively investigated because of its impact on plant growth and yield. So far, the effects of a combined nutrient limitation have rarely been analyzed, although such situations are likely to occur in agroecosystems. Iron (Fe) is a prerequisite for many essential cellular functions. Its availability is easily becoming limiting for plant growth and thus higher plants have evolved different strategies to cope with Fe deficiency. Sulfur (S) is an essential macro-nutrient and the responses triggered by shortage situations have been well characterized. The interaction between these two nutrients is less investigated but might be of particular importance because most of the metabolically active Fe is bound to S in Fe-S clusters. The biosynthesis of Fe-S clusters requires the provision of reduced S and chelated Fe in a defined stoichiometric ratio, strongly suggesting coordination between the metabolisms of the two nutrients. Here the available information on interactions between Fe and S nutritional status is evaluated. Experiments with Arabidopsis thaliana and crop plants indicate a co-regulation and point to a possible role of Fe-S cluster synthesis or abundance in the Fe/S network.
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Sauter M, Moffatt B, Saechao MC, Hell R, Wirtz M. (2013).
Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis.
Biochem J. 451: 145-154. 
Abstract
Both Met (methionine) and SAM (S-adenosylmethionine), the activated form of Met, participate in a number of essential metabolic pathways in plants. The subcellular compartmentalization of Met fluxes will be discussed in the present review with respect to regulation and communication with the sulfur assimilation pathway, the network of the aspartate-derived amino acids and the demand for production of SAM. SAM enters the ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for the majority of methylation reactions required for plant growth and development. The multiple essential roles of SAM require regulation of its synthesis, recycling and distribution to sustain these different pathways. A particular focus of this review will be on the function of recently identified genes of the Met salvage cycle or Yang cycle and the importance of the Met salvage cycle in the metabolism of MTA(5-methylthioadenosine). MTA has the potential for product inhibition of ethylene, nicotianamine and polyamine biosynthesis which provides an additional link between these pathways. Interestingly, regulation of Met cycle genes was found to differ between plant species as shown for Arabidopsis thaliana and Oryza sativa.
Pubmed 
Chan KX, Wirtz M, Phua SY, Estavillo GM, Pogson BJ. (2013).
Balancing metabolites in drought: the sulfur assimilation conundrum.
Trends Plant Sci. 18: 18 - 29. 
Abstract
A key plant response to drought is the accumulation of specific sets of metabolites that act as osmoprotectants, osmolytes, antioxidants, and/or stress signals. An emerging question is: how do plants regulate metabolism to balance the 'competing interests' between metabolites during stress? Recent research connects primary sulfur metabolism (e.g., sulfate transport in the vasculature, its assimilation in leaves, and the recycling of sulfur-containing compounds) with the drought stress response. In this review, we highlight key steps in sulfur metabolism that play significant roles in drought stress signaling and responses. We propose that a complex balancing act is required to coordinate primary and secondary sulfur metabolism during the drought stress response in plants.
Pubmed 


2012

Van de Poel B, Bulens I, Markoula A, Hertog M, Dreesen R, Wirtz M, Vandoninck S, Oppermann Y, Keulemans W, Hell R, Waelkens E, De Proft MP, Sauter M, Nicolai BM, Geeraerd AH. (2012).
Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during post-climacteric ripening.
Plant Physiol. 160: 1498 - 1514. 
Abstract
The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the post-climacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. At the same time ACC synthase (ACS) activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during post-climacteric ripening. Post-climacteric red tomatoes on the plant showed only a moderate regulation of ACS and Yang cycle genes compared to the regulation in detached fruit. Treatment of red fruit with 1-MCP and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene synthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.
Pubmed 
Schiavon M, Galla G, Wirtz M, Pilon-Smits EA, Telatin V, Quaggiotti S, Hell R, Barcaccia G, Malagoli M. (2012).
Transcriptome profiling of genes differentially modulated by sulfur and chromium identifies potential targets for phytoremediation and reveals a complex S-Cr interplay on sulfate transport regulation in B. juncea.
J Hazard Mater. 239-240: 192-205. 
Abstract
A differential display cDNA-AFLP derived technique was used to identify gene transcripts regulated by chromium (Cr) in relation to sulfur (S) nutrition in Brassica juncea. Twelve-day old plants were grown with 200?M sulfate (+S), without sulfate (-S), with 200?M sulfate plus 200?M chromate (+S+Cr), or without sulfate plus 200?M chromate (-S+Cr). Forty-four combinations of degenerate primers were assayed, which allowed the detection of 346 Transcript-Derived Fragments (TDFs) differentially regulated by Cr and S at times 0, 10min, 1h, 24h. Eight sulfate transporters were identified, whose transcript abundance was dependent on the levels of plant S-compounds. For some of these transporters, a tight coordinated regulation of gene expression was observed in response to Cr. The MapMan analysis revealed a differential pattern of gene expression between +S+Cr and -S+Cr plants for several other transcripts and highlighted an overlap among responses to metals, defence against pathogens and senescence, hence suggesting the existence of common mechanisms of gene regulation. Among the identified gene transcripts, those involved in S metabolism and proteolitic processes may represent potential targets of genetic engineering in efforts to increase Cr accumulation and tolerance in plant species employed in phytoremediation techniques.
Pubmed 
Birke H, Haas FH, De Kok LJ, Balk J, Wirtz M, Hell R. (2012).
Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana.
Biochem J.  445: 275-283.
Abstract
In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by β-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.
Pubmed 
Birke H, Müller SJ, Rother M, Zimmer AD, Hoernstein SN, Wesenberg D, Wirtz M, Krauss GJ, Reski R, Hell R. (2012).
The relevance of compartmentation for cysteine synthesis in phototrophic organisms.
Protoplasma  249: 147-155
Abstract
In the vascular plant Arabidopsis thaliana, synthesis of cysteine and its precursors O-acetylserine and sulfide is distributed between the cytosol, chloroplasts, and mitochondria. This compartmentation contributes to regulation of cysteine synthesis. In contrast to Arabidopsis, cysteine synthesis is exclusively restricted to chloroplasts in the unicellular green alga Chlamydomonas reinhardtii. Thus, the question arises, whether specification of compartmentation was driven by multicellularity and specified organs and tissues. The moss Physcomitrella patens colonizes land but is still characterized by a simple morphology compared to vascular plants. It was therefore used as model organism to study evolution of compartmented cysteine synthesis. The presence of O-acetylserine(thiol)lyase (OAS-TL) proteins, which catalyze the final step of cysteine synthesis, in different compartments was applied as criterion. Purification and characterization of native OAS-TL proteins demonstrated the presence of five OAS-TL protein species encoded by two genes in Physcomitrella. At least one of the gene products is dual targeted to plastids and cytosol, as shown by combination of GFP fusion localization studies, purification of chloroplasts, and identification of N termini from native proteins. The bulk of OAS-TL protein is targeted to plastids, whereas there is no evidence for a mitochondrial OAS-TL isoform and only a minor part of OAS-TL protein is localized in the cytosol. This demonstrates that subcellular diversification of cysteine synthesis is already initialized in Physcomitrella but appears to gain relevance later during evolution of vascular plants.
Pubmed 
Bok-Rye L, Huseby S, Koprivova A, Chetelat A, Wirtz M, Mugford ST, Navid E, Brearley C, Saha S, Mithen R, Hell R, Farmer EE, Kopriva S (2012).
Effects of fou8/fry1 Mutation on Sulfur Metabolism: Is Decreased Internal Sulfate the Trigger of Sulfate Starvation Response?
PLoS ONE.  7 (6): e39425.
Abstract
The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5-phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3-phosphoadenosine 5-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism.
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Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Krämer U. (2012).
Vacuolar Nicotianamine Has Critical and Distinct Roles under Iron Deficiency and for Zinc Sequestration in Arabidopsis.
Plant Cell. 24: 724-737
Abstract
The essential micronutrients Fe and Zn often limit plant growth but are toxic in excess. Arabidopsis thaliana ZINC-INDUCED FACILITATOR1 (ZIF1) is a vacuolar membrane major facilitator superfamily protein required for basal Zn tolerance. Here, we show that overexpression of ZIF1 enhances the partitioning into vacuoles of the low molecular mass metal chelator nicotianamine and leads to pronounced nicotianamine accumulation in roots, accompanied by vacuolar buildup of Zn. Heterologous ZIF1 protein localizes to vacuolar membranes and enhances nicotianamine contents of yeast cells engineered to synthesize nicotianamine, without complementing a Zn-hypersensitive mutant that additionally lacks vacuolar membrane Zn(2+)/H(+) antiport activity. Retention in roots of Zn, but not of Fe, is enhanced in ZIF1 overexpressors at the expense of the shoots. Furthermore, these lines exhibit impaired intercellular Fe movement in leaves and constitutive Fe deficiency symptoms, thus phenocopying nicotianamine biosynthesis mutants. Hence, perturbing the subcellular distribution of the chelator nicotianamine has profound, yet distinct, effects on Zn and Fe with respect to their subcellular and interorgan partitioning. The zif1 mutant is also hypersensitive to Fe deficiency, even in media lacking added Zn. Therefore, accurate levels of ZIF1 expression are critical for both Zn and Fe homeostasis. This will help to advance the biofortification of crops.
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Kruse C, Haas FH, Jost R, Reiser B, Reichelt M, Wirtz M, Gershenzon J, Schnug E, Hell R. (2012).
Improved sulfur nutrition provides the basis for enhanced production of sulfur-containing defense compounds in Arabidopsis thaliana upon inoculation with Alternaria brassicicola.
J Plant Physiol.169: 740-743
Abstract
The antifungal activities of many sulfur-containing defense compounds suggest a connection between pathogen infection, primary sulfur metabolism and sulfate nutritional status of plants. This relationship was investigated using Arabidopsis thaliana plants that were cultivated under different sulfur regimes and challenged by Alternaria brassicicola. Plants grown with 500μM sulfate were significantly less infected compared to plants grown on 50μM sulfate. Upon infection, the formation of the sulfur-containing defense compound camalexin and the gene expression of the sulfur-rich defense peptide defensin were clearly enhanced in plants grown with an optimal compared to a sufficient sulfate supply in the growth medium. Elevated levels of sulfite and O-acetylserine and cysteine biosynthetic enzymes after infection indicated a stimulation of sulfur metabolism under the higher sulfate supply. The results suggest that, in addition to pathogen-triggered activation of sulfur metabolism and sulfur-containing defense compound formation, the sulfate nutritional status is sensed to contribute to plant defense.
Pubmed 
Feldman-Salit A, Wirtz M, Lenherr ED, Throm C, Hothorn M, Scheffzek K, Hell R, Wade RC. (2012).
Allosterically Gated Enzyme Dynamics in the Cysteine Synthase Complex Regulate Cysteine Biosynthesis in Arabidopsis thaliana.
Structure. 20:292-302.
Abstract
Plants and bacteria assimilate sulfur into cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase complex (CSC), which consists of serine-acetyl-transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL) enzymes. The activity of OAS-TL is reduced by formation of the CSC. Although this reduction is an inherent part of the self-regulation cycle of cysteine biosynthesis, there has until now been no explanation as to how OAS-TL loses activity in plants. Complexation of SAT and OAS-TL involves binding of the C-terminal tail of SAT in one of the active sites of the homodimeric OAS-TL. We here explore the flexibility of the unoccupied active site in Arabidopsis thaliana cytosolic and mitochondrial OAS-TLs. Our results reveal two gates in the OAS-TL active site that define its accessibility. The observed dynamics of the gates show allosteric closure of the unoccupied active site of OAS-TL in the CSC, which can hinder substrate binding, abolishing its turnover to cysteine.
Pubmed 
Schiavon, M., Pittarello, M., Pilon-Smits, E.A.H., Wirtz, M., Hell, R., Malagoli, M. (2012).
Selenate and molybdate alter sulfate transport and assimilation in Brassica juncea L. Czern.: Implications for phytoremediation.
Environmental and Experimental Botany. 75: 41–51.
Abstract
The interaction of selenate and molybdate with the transport and assimilation of sulfate, and the effect of S on Se and Mo accumulation were investigated in Brassica juncea. Plants were supplied with different combinations of S and Se, or S and Mo for 24 h, and selenate and molybdate were given to plants at concentrations (200 μM) equal to that of sulfate in the S-sufficient condition. Se and Mo significantly reduced the plant growth. In S-sufficient plants, Se and Mo decreased sulfate uptake rate (at 24 h), and Se repressed the expression of the sulfate transporter BjSultr2.1. This effect was different from the one we observed for sulfate, which rapidly inhibited the sulfate uptake rates in +S plants. In S-starved plants, Se, Mo and S repressed sulfate uptake immediately (after 10 min), and the concomitant down-regulation of BjSultr2.1occurred only in roots of plants treated with S. In plants exposed to Se or Mo the root expression of BjSultr2.1was repressed later, after 6 h. S-starved plants accumulated significantly more Se and Mo than S-sufficient plants, likely due to the lack of competition of molybdate or selenate with sulfate for the transport through the same carriers. The up-regulation of the molybdenum transporter (MOT1) gene expression could explain the higher amount of Mo than Se measured in the plant tissues. Se and Mo reduced the levels of cysteine (Cys) and glutathione (GSH) in +S plants, but increased the amount of these non-protein thiols in −S plants. The increase of GSH content in −S + Se plants was likely responsible for the down-regulation of the selenium binding protein (SBP1) gene, while the induction of SBP1observed in +S plants was mainly due to Se toxicity. The up-regulation of SBP1 was also evidenced in plants exposed to Mo, regardless of S availability and GSH content. Our results give better insight into plant uptake mechanisms for Se and Mo, and also have implications for phytoremediation. The interactions between sulfate and selenate or molybdate must be carefully considered when plants are employed for the remediation of Se- or Mo-contaminated sites, as the accumulation of the two contaminants in plants might be altered by the sulfate concentration in the growing medium.
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Waduwara-Jayabahu I, Oppermann Y, Wirtz M, Hull ZT, Schoor S, Plotnikov AN, Hell R, Sauter M, Moffatt BA. (2012).
Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis.
Plant Physiol.158: 1728-1744
Abstract
5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.
Pubmed 
Wirtz M, Beard KFM, Lee CP, Boltz A, Schwarzlaender M, Fuchs C, Meyer AJ, Heeg C, Sweetlove LJ, Ratcliffe RG Hell R. (2012).
Mitochondrial cysteine synthase complex regulates O-acetylserine biosynthesis in plants
J Biol Chem.  287: 27941-27947.
Abstract
Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by Foerster resonance energy transfer (FRET) and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. Nuclear magnetic resonance (NMR) spectroscopy of isolated mitochondria from wild type, serat2;2 and oastl-C plants shows the SAT-dependent export of O-acetylserine (OAS). The presence of cysteine results in reduced OAS export in mitochondria of oastl-C mutants but not of wildtype. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine as compared to CSC bound SAT and explains the high OAS export rate of wild type mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis is validated in planta by feeding 3H-labeled serine to wild type and loss-of-function mutants for OAS-TLs in the cytosol, the plastids and the mitochondria. Based on these results we propose a new model, in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.
Pubmed 


2011

Wirtz M, Hell R. (2011).
Molecular Biology, Biochemistry and Cellular Physiology of Cysteine Metabolism in Arabidopsis thaliana.
The Arabidopsis Book. 9: e0154.2011.
Abstract
Cysteine is one of the most versatile molecules in biology, taking over such different functions as catalysis, structure, regulation and electron transport during evolution. Research on Arabidopsis has contributed decisively to the understanding of cysteine synthesis and its role in the assimilatory pathways of S, N and C in plants. The multimeric cysteine synthase complex is present in the cytosol, plastids and mitochondria and forms the centre of a unique metabolic sensing and signaling system. Its association is reversible, rendering the first enzyme of cysteine synthesis active and the second one inactive, and vice-versa. Complex formation is triggered by the reaction intermediates of cysteine synthesis in response to supply and demand and gives rise to regulation of genes of sulfur metabolism to adjust cellular sulfur homeostasis. Combinations of biochemistry, forward and reverse genetics, structural- and cell-biology approaches using Arabidopsis have revealed new enzyme functions and the unique pattern of spatial distribution of cysteine metabolism in plant cells. These findings place the synthesis of cysteine in the centre of the network of primary metabolism.
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Estavillo GM, Crisp PA, Pornsiriwong W, Wirtz M, Collinge D, Carrie C, Giraud E, Whelan J, David P, Javot H, Brearley C, Hell R, Marin E, Pogson BJ. (2011).
Evidence for a SAL1-PAP Chloroplast Retrograde Pathway That Functions in Drought and High Light Signaling in Arabidopsis.
Plant Cell.  23:3992-4012
Abstract 
Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.
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Hsu FC, Wirtz M, Heppel SC, Bogs J, Krämer U, Khan MS, Bub A, Hell R, Rausch T. (2011).
Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism.
Plant Cell Environ. 34:192-207. 
Abstract
Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.
Pubmed 


2010

Wirtz M, Birke H, Heeg C, Müller C, Hosp F, Throm C, König S, Feldman-Salit A, Rippe K, Petersen G, Wade RC, Rybin V, Scheffzek K, Hell R. (2010).
Structure and function of the hetero-oligomeric cysteine synthase complex in plants.
J Biol Chem. 285:32810-7. 
Abstract
Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(D) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(D)) and the regulation of cysteine sensitivity of SAT within the CSC.
Pubmed 
Zuber H, Davidian JC, Aubert G, Aimé D, Belghazi M, Lugan R, Heintz D, Wirtz M, Hell R, Thompson R, Gallardo K. (2010).
The seed composition of Arabidopsis mutants for the group 3 sulfate transporters indicates a role in sulfate translocation within developing seeds.
Plant Physiol. 154:913-26. 
Abstract
Sulfate is required for the synthesis of sulfur-containing amino acids and numerous other compounds essential for the plant life cycle. The delivery of sulfate to seeds and its translocation between seed tissues is likely to require specific transporters. In Arabidopsis (Arabidopsis thaliana), the group 3 plasmalemma-predicted sulfate transporters (SULTR3) comprise five genes, all expressed in developing seeds, especially in the tissues surrounding the embryo. Here, we show that sulfur supply to seeds is unaffected by T-DNA insertions in the SULTR3 genes. However, remarkably, an increased accumulation of sulfate was found in mature seeds of four mutants out of five. In these mutant seeds, the ratio of sulfur in sulfate form versus total sulfur was significantly increased, accompanied by a reduction in free cysteine content, which varied depending on the gene inactivated. These results demonstrate a reduced capacity of the mutant seeds to metabolize sulfate and suggest that these transporters may be involved in sulfate translocation between seed compartments. This was further supported by sulfate measurements of the envelopes separated from the embryo of the sultr3;2 mutant seeds, which showed differences in sulfate partitioning compared with the wild type. A dissection of the seed proteome of the sultr3 mutants revealed protein changes characteristic of a sulfur-stress response, supporting a role for these transporters in providing sulfate to the embryo. The mutants were affected in 12S globulin accumulation, demonstrating the importance of intraseed sulfate transport for the synthesis and maturation of embryo proteins. Metabolic adjustments were also revealed, some of which could release sulfur from glucosinolates.
Pubmed 
Wirtz M, Heeg C, Samami AA, Ruppert T, Hell R. (2010).
Enzymes of cysteine synthesis show extensive and conserved modifications patterns that include N(α)-terminal acetylation.
Amino Acids. 39:1077-86. 
Abstract
Biosynthesis of cysteine is a two-step process in higher plants subsequently catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which are present in cytosol, plastids and mitochondria. Recently, the distribution of SAT and OAS-TL in these subcellular compartments was shown to be crucial for efficient cysteine synthesis in Arabidopsis thaliana. In this study, the abundances of OAS-TLs were quantified independently by immunological detection in crude protein extracts and by SAT affinity purification (SAP) of OAS-TL. OAS-TL A and B were evidenced to be the most abundant isoforms in all analyzed tissues, which is consistent with micro array-based transcript analyses. Application of SAP to Arabidopsis revealed significant modification of the major OAS-TL isoforms present in cytosol, plastids and mitochondria into up to seven subspecies. Specific OAS-TL isoforms were found to be differentially modified in the leaves, roots, stem and cell culture. Sulphur deficiency did not alter modification of OAS-TL proteins purified from cell culture that showed the highest complexity of OAS-TL modifications. However, the pattern of OAS-TL modification was found to be stable within an analyzed tissue, pointing not only to a high reproducibility of SAP but likely biological significance of each subspecies. The most abundant OAS-TL subspecies in cytosol and plastids were subject of N-terminal processing followed by acetylation of the newly originated N-terminus. The mode of N(α)-terminal acetylation of OAS-TL and its possible biological function are discussed.
Pubmed 
Zuber H, Davidian JC, Wirtz M, Hell R, Belghazi M, Thompson R, Gallardo K. (2010).
Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization.
BMC Plant Biol. 10:78. 
Abstract
BACKGROUND: Sulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole. RESULTS: In the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes. CONCLUSIONS: This study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.
Pubmed 
Khan MS, Haas FH, Samami AA, Gholami AM, Bauer A, Fellenberg K, Reichelt M, Hänsch R, Mendel RR, Meyer AJ, Wirtz M, Hell R. (2010).
Sulfite reductase defines a newly discovered bottleneck for assimilatory sulfate reduction and is essential for growth and development in Arabidopsis thaliana.
Plant Cell. 22:1216-31. 
Abstract
The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of (35)S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.
Pubmed 
Bürstenbinder K, Waduwara I, Schoor S, Moffatt BA, Wirtz M, Minocha SC, Oppermann Y, Bouchereau A, Hell R, Sauter M. (2010).
Inhibition of 5'-methylthioadenosine metabolism in the Yang cycle alters polyamine levels, and impairs seedling growth and reproduction in Arabidopsis.
Plant J. 62:977-88. 
Abstract
The methionine or Yang cycle recycles Met from 5'-methylthioadenosine (MTA) which is produced from S-adenosyl-L-methionine (SAM) as a by-product of ethylene, polyamines, and nicotianamine (NA) synthesis. MTA nucleosidase is encoded by two genes in Arabidopsis thaliana, MTN1 and MTN2. Analysis of T-DNA insertion mutants and of wt revealed that MTN1 provides approximately 80% of the total MTN activity. Severe knock down of MTN enzyme activity in the mtn1-1 and mtn1-2 allelic lines resulted in accumulation of SAM/dSAM (decarboxylated SAM) and of MTA in seedlings grown on MTA as sulfur source. While ethylene and NA synthesis were not altered in mtn1-1 and mtn1-2 seedlings grown on MTA, putrescine and spermine were elevated. By contrast, mtn2-1 and mtn2-2 seedlings with near wt enzyme activity had wt levels of SAM/dSAM, MTA, and polyamines. In addition to the metabolic phenotypes, mtn1-1 and mtn1-2 seedlings were growth retarded, while seedlings of wt, mtn2-1, and mtn2-2 showed normal growth on 500 microm MTA. The double knock down mutant mtn1-1/mtn2-1 was sterile. In conclusion, the data presented identify MTA as a crucial metabolite that acts as a regulatory link between the Yang cycle and polyamine biosynthesis and identifies MTA nucleosidase as a crucial enzyme of the Yang cycle.
Pubmed 


2009

Tabe L, Wirtz M, Molvig L, Droux M, Hell R. (2009).
Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume.
J Exp Bot. 61:721-33. 
Abstract
There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosynthesis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F(2) seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein.
Pubmed 
Bräutigam K, Dietzel L, Kleine T, Ströher E, Wormuth D, Dietz KJ, Radke D, Wirtz M, Hell R, Dörmann P, Nunes-Nesi A, Schauer N, Fernie AR, Oliver SN, Geigenberger P, Leister D, Pfannschmidt T. (2009).
Dynamic plastid redox signals integrate gene expression and metabolism to induce distinct metabolic states in photosynthetic acclimation in Arabidopsis.
Plant Cell. 21:2715-32. 
Abstract
Plants possess acclimation responses in which structural reconfigurations adapt the photosynthetic apparatus to fluctuating illumination. Long-term acclimation involves changes in plastid and nuclear gene expression and is controlled by redox signals from photosynthesis. The kinetics of these signals and the adjustments of energetic and metabolic demands to the changes in the photosynthetic apparatus are currently poorly understood. Using a redox signaling system that preferentially excites either photosystem I or II, we measured the time-dependent impact of redox signals on the transcriptome and metabolome of Arabidopsis thaliana. We observed rapid and dynamic changes in nuclear transcript accumulation resulting in differential and specific expression patterns for genes associated with photosynthesis and metabolism. Metabolite pools also exhibited dynamic changes and indicate readjustments between distinct metabolic states depending on the respective illumination. These states reflect reallocation of energy resources in a defined and reversible manner, indicating that structural changes in the photosynthetic apparatus during long-term acclimation are additionally supported at the level of metabolism. We propose that photosynthesis can act as an environmental sensor, producing retrograde redox signals that trigger two parallel adjustment loops that coordinate photosynthesis and metabolism to adapt plant primary productivity to the environment.
Pubmed 
Marty L, Siala W, Schwarzländer M, Fricker MD, Wirtz M, Sweetlove LJ, Meyer Y, Meyer AJ, Reichheld JP, Hell R. (2009).
The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis.
Proc Natl Acad Sci U S A. 106:9109-14. 
Abstract
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.
Pubmed 
Roeder S, Dreschler K, Wirtz M, Cristescu SM, van Harren FJ, Hell R, Piechulla B. (2009).
SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers.
Plant Mol Biol. 70:535-46. 
Abstract
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).
Pubmed 
Mugford SG, Yoshimoto N, Reichelt M, Wirtz M, Hill L, Mugford ST, Nakazato Y, Noji M, Takahashi H, Kramell R, Gigolashvili T, Flügge UI, Wasternack C, Gershenzon J, Hell R, Saito K, Kopriva S. (2009).
Disruption of adenosine-5'-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
Plant Cell. 21:910-27. 
Abstract
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.
Pubmed 
Klatte M, Schuler M, Wirtz M, Fink-Straube C, Hell R, Bauer P. (2009).
The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses.
Plant Physiol. 150:257-71. 
Abstract
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.
Pubmed 


2008

Feldman-Salit A, Wirtz M, Hell R, Wade RC. (2008).
A mechanistic model of the cysteine synthase complex.
J Mol Biol. 386:37-59. 
Abstract
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.
Pubmed 
Haas FH, Heeg C, Queiroz R, Bauer A, Wirtz M, Hell R. (2008).
Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells.
Plant Physiol. 148:1055-67. 
Abstract
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.
Pubmed 
Schiavon M, Pilon-Smits EA, Wirtz M, Hell R, Malagoli M. (2008).
Interactions between chromium and sulfur metabolism in Brassica juncea.
J Environ Qual. 37:1536-45. 
Abstract
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.
Pubmed 
Rouached H, Wirtz M, Alary R, Hell R, Arpat AB, Davidian JC, Fourcroy P, Berthomieu P. (2008).
Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis.
Plant Physiol. 147:897-911. 
Abstract
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.
Pubmed 
Heeg C, Kruse C, Jost R, Gutensohn M, Ruppert T, Wirtz M, Hell R. (2008).
Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene family demonstrates compartment-specific differences in the regulation of cysteine synthesis.
Plant Cell. 20:168-85. 
Abstract
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.
Pubmed 


2007

Pasternak M, Lim B, Wirtz M, Hell R, Cobbett CS, Meyer AJ. (2007).
Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development.
Plant J. 53:999-1012. 
Abstract
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.
Pubmed 
Schiavon M, Wirtz M, Borsa P, Quaggiotti S, Hell R, Malagoli M. (2007).
Chromate differentially affects the expression of a high-affinity sulfate transporter and isoforms of components of the sulfate assimilatory pathway in Zea mays (L.).
Plant Biol 9:662-71. 
Abstract
In this study the chromate accumulation and tolerance were investigated in ZEA MAYS L. in relation to sulfur availability since sulfate may interact with chromate for transport into the cells. Chromate inhibited sulfate uptake when supplied to plants for a short-term period, whereas phosphate uptake remained unchanged. Sulfate absorption was also reduced in S-starved (-S) and S-supplied (+S) plants treated for 2 d with 0.2 mM chromate and the concomitant repression of the root high-affinity sulfate root transporter ZMST1;1 transcript accumulation was observed. Conversely, the plasma membrane H (+)-ATPase MHA2 was unaffected by chromate in +S plants, allowing to exclude a general effect of chromate on the active membrane transport. As observed for sulfate uptake, chromate uptake was enhanced in -S condition and decreased in both +S and -S plants after 2 d of Cr treatment. Chromate reduced the concentration of sulfur and sulfate in +S plants to the basal level of -S plants, and maximum chromium accumulation was recorded in S-deprived plants. Analysis of transcript abundance of genes involved in sulfate assimilation revealed differential regulation by chromate, which was only partly related to sulfur availability and to the levels of thiols. This work shows for the first time that chromate specifically represses sulfate uptake, and such repression occurs without the implication of the candidate regulatory metabolites of the sulfate transport system in plants.
Pubmed 
Rzewuski G, Cornell KA, Rooney L, Bürstenbinder K, Wirtz M, Hell R, Sauter M. (2007).
OsMTN encodes a 5'-methylthioadenosine nucleosidase that is up-regulated during submergence-induced ethylene synthesis in rice (Oryza sativa L.).
J Exp Bot. 58:1505-14. 
Abstract
Methylthioadenosine (MTA) is released as a by-product of S-adenosylmethionine (AdoMet)-dependent reactions central to ethylene, polyamine, or phytosiderophore biosynthesis. MTA is hydrolysed by methylthioadenosine nucleosidase (MTN; EC 3.2.2.16) into adenine and methylthioribose which is processed through the methionine (Met) cycle to produce a new molecule of AdoMet. In deepwater rice, submergence enhances ethylene biosynthesis, and ethylene in turn influences the methionine cycle through positive feedback regulation of the acireductone dioxygenase gene OsARD1. In rice, MTN is encoded by a single gene designated OsMTN. Recombinant OsMTN enzyme had a KM for MTA of 2.1 mM and accepted a wide array of 5' substitutions of the substrate. OsMTN also metabolized S-adenosylhomocysteine (AdoHcy) with 15.9% the rate of MTA. OsMTN transcripts and OsMTN-specific activity increased slowly and in parallel upon submergence, indicating that regulation occurred mainly at the transcriptional level. Neither ethylene, MTA, nor Met regulated OsMTN expression. Analysis of steady-state metabolite levels showed that MTN activity was sufficiently high to prevent Met and AdoMet depletion during long-term ethylene biosynthesis.
Pubmed 
Lang C, Popko J, Wirtz M, Hell R, Herschbach C, Kreuzwieser J, Rennenberg H, Mendel RR, Hänsch R. (2007).
Sulphite oxidase as key enzyme for protecting plants against sulphur dioxide.
Plant Cell Environ. 30:447-55. 
Abstract
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.
Pubmed 
Wirtz M, Hell R. (2007).
Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco.
Plant Cell. 19:625-39. 
Abstract
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.
Pubmed 


2006

Bürstenbinder K, Rzewuski G, Wirtz M, Hell R, Sauter M. (2006).
The role of methionine recycling for ethylene synthesis in Arabidopsis.
Plant J. 49:238-49. 
Abstract
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.
Pubmed 


2005

Wirtz M, Hell R. (2005).
Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties.
J Plant Physiol. 163:273-86. 
Abstract
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.
Pubmed 
Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hähnel U, Hänsch R, Hartmann T, Kopriva S, Kruse C, Mendel RR, Papenbrock J, Reichelt M, Rennenberg H, Schnug E, Schmidt A, Textor S, Tokuhisa J, Wachter A, Wirtz M, Rausch T, Hell R.(2005).
Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana.
Photosynth Res. 86:491-508. 
Abstract
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.
Pubmed 


2004

Fey V, Wagner R, Braütigam K, Wirtz M, Hell R, Dietzmann A, Leister D, Oelmüller R, Pfannschmidt T. (2004).
Retrograde plastid redox signals in the expression of nuclear genes for chloroplast proteins of Arabidopsis thaliana.
J Biol Chem. 280:5318-28. 
Abstract
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.
Pubmed 
Buchner P, Stuiver CE, Westerman S, Wirtz M, Hell R, Hawkesford MJ, De Kok LJ. (2004).
Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition.
Plant Physiol. 136:3396-408. 
Abstract
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.
Pubmed 
Wirtz M, Droux M, Hell R. (2004).
O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana.
J Exp Bot. 55:1785-98. 
Abstract
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)
Pubmed 
Hartmann T, Hönicke P, Wirtz M, Hell R, Rennenberg H, Kopriva S. (2004).
Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol.
J Exp Bot. 55:837-45. 
Abstract
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.
Pubmed 


2003

Wirtz M, Hell R. (2003).
Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase.
Amino Acids 24:195-203. 
Abstract
The first step of cysteine biosynthesis in bacteria and plants consists in the formation of O-acetylserine catalyzed by serine acetyltransferase (SAT). SAT is highly sensitive to feedback inhibition by cysteine as part of the regulatory circuit of cysteine biosynthesis und thus hampers over-expression and fermentation of cysteine in biotechnological production processes. Since plants contain multiple SAT isoforms with different cysteine feedback sensitivity, this resource was exploited to demonstrate the suitability of plant SATs for the production of cysteine in both bacteria and plants. Three new cDNAs encoding SATs were isolated from Nicotiana tabacum. The catalytic activity of SAT4 was insensitive up to 0.6 mM cysteine. Expression of SAT4 in a newly constructed Escherichia coli host strain without endogenous SAT activity yielded a significant accumulation of cysteine in the culture medium compared to expression of cysteine sensitive SATs in the same strain. The application of a similarly insensitive SAT isoform from A. thaliana demonstrated the suitability of this approach to increase cysteine levels in transgenic tobacco plants.
Pubmed 


2002

Hell R, Jost R, Berkowitz O, Wirtz M. (2002).
Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant Arabidopsis thaliana.
Amino Acids 22:245-57. 
Abstract
Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur for cell metabolism. Part of this characteristic feature is the presence of cysteine synthesis in plastids, mitochondria and cytosol. Plants are the major source of reduced sulfur for human and animal nutrition. Cysteine biosynthesis deserves special attention, since reduced sulfur is channelled from cysteine into many sulfur-containing compounds in food and feed. Recent investigations are reviewed that focus on structure and regulation of cysteine synthesis in the model plant Arabidopsis thaliana. These data indicate that cysteine synthesis is not just an intermediate reaction step but that it is part of a regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes.
Pubmed 
Berkowitz O, Wirtz M, Wolf A, Kuhlmann J, Hell R. (2002).
Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana.
J Biol Chem. 277:30629-34. 
Abstract
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 + BAB 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.
Pubmed 


2001

Wirtz M, Berkowitz O, Droux M, Hell R. (2001).
The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.
Eur J Biochem. 268:686-93. 
Abstract
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.
Pubmed 


2000

Jost R, Berkowitz O, Wirtz M, Hopkins L, Hawkesford MJ, Hell R. (2000).
Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana.
Gene. 253:237-47. 
Abstract
The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.
Pubmed 


/var/www/cos/ / http://www.cos.uni-heidelberg.de/ Dr. Markus Wirtz