Ruprecht-Karls-Universität Heidelberg
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Plant Molecular Biology

Prof. Dr. Rüdiger Hell

 

2017

Eva-Sophie Wallner, E.-S., López-Salmerón, V., Belevich, I., Poschet, G., Jung, I., Grünwald, K., Sevilem, Jokitalo, E., Hell, R., Helariutta, Y., Agustí, J., Lebovka, I., Greb, T. (2017) SL/KAR-independent SMXL proteins are central regulators of phloem formation. Curr. Biol., accepted

Giaretta, S., Prasad, D., Forieri, I., Vamerali, T., Trentin, A. R., Wirtz; M., Hell, R., Masi, A. (2017) Apoplastic activity of GGT encoded by GGT1 and GGT2 is important for vegetative and generative development. Plant Physiol. Biochem., accepted

2016

Weger, B. D., Weger, M., Görling, B., Schink, A., Gobet, C., Keime, C., Poschet, G., Jost, B., Krone, N., Hell, R., Gachon, F., Luy, B., Dickmeis, T. (2016) Extensive regulation of diurnal transcription and metabolism by glucocorticoids. PLoS Genetics, 12:12, e1006512. doi: 10.1371/journal.pgen.1006512

Kovacs, I., Holzmeister, C., Wirtz, M., Geerlof, A., Fröhlich, T., Römling, G., Kuruthukulangarakoola, T.G., Linster, E., Hell, R., Arnold, G.J., Durner, J., Lindermayr, C., (2016) ROS-mediated inhibition of S-nitrosoglutathione reductase contributes to the activation of anti-oxidative mechanisms. Front. Plant Sci., 10;7:1669, DOI: 10.3389/fpls.2016.01669

Ahmad, N., Malagoli, M., Wirtz, M., Hell, R. (2016) Drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots. BMC Plant Biology 16: 247, DOI: 10.1186/s12870-016-0940-z

Forieri, I., Sticht, C., Reichelt, M., Gretz, N., Hawkesford, M.J., Malagoli, M., Wirtz, M., Hell, R. (2017) Systems analysis of metabolism and the transcriptome in Arabidopsis thaliana roots reveals differential co-regulation upon iron, sulfur and potassium deficiency. Plant, Cell Environm. 40: 95–107. DOI: 10.1111/pce.12842

Huang, L.-J., Ning Li, N., Thurow, C., Wirtz, M., Hell, R., Gatz, C. (2016) Ectopically expressed glutaredoxin ROXY19 negatively regulates the detoxification pathway in Arabidopsis thaliana. BMC Plant Biology, doi: 10.1186/s12870-016-0886-1

Huang, X.-Y., Chao, D.-Y., Koprivova, A., Danku, J., Wirtz, M., Müller, S., Sandoval, F.J., Bauwe, H., Roje, S., Dilkes, B., Hell, R., Kopriva, S., Salt, D.E. (2016) Nuclear localised MORE SULPHUR ACCUMULATION1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana. PLoS Genet 12: e1006298. doi:10.1371/journal.pgen.1006298

Lee, C., Maksaev, G., Jensen, G., Murcha, M., Wilson, M., Fricker, M., Hell, R., Haswell, E., Millar, A. H., Sweetlove, L. (2016) MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress. Plant J. 88: 809–825, DOI: 10.1111/tpj.13301

Pfeiffer, A., Janocha, D., Dong, Y., Medzihradszky, A., Schöne,S., Daum, G., Suzaki, T., Forner, J., Langenecker, T., Schmid, M., Wirtz, M., Hell, R., Lohmann, J. U. (2016) Integration of light and metabolic signals for stem cell activation at the shoot apical meristem. eLife, doi: 10.7554/eLife.17023

Groth, M., Moissiard, G., Wirtz, M., Wang, H., Garcia-Salinas, C., Ramos-Parra, P. A., Bischof, S., Feng, S., Cokus, S.J., John, A., Smith, D. C., Zhai, J., Hale, C. J., Long, J. A., Hell, R., Díaz de la Garza, R. I., Jacobsen, S. E. (2016) MTHFD1 controls DNA methylation in Arabidopsis. Nature Comm. 7: 11640. doi: 10.1038/ncomms11640

Kopriva, S., Talukdar, D., Takahashi, H., Hell, R., Sirko, A., D’Souza, S.F., Talukdar, T. (2016) Editorial: Frontiers of sulfur metabolism in plant growth, development and stress response. Front. Plant Sci., 6: 1220. doi:10.3389/fpls.2015.01220

 

 

 

2015

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, 27:1968-1984

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 Nα-acetyltransferase in plastids by global acetylome profiling. Proteomics, 15:2426-2435, doi: 10.1002/pmic.201500025

Linster, E., Stephan, I., Bienvenut, W.V., Maple-Grødem, J., Myklebust, L.M., Huber, M., Reichelt, M., Sticht, C., Møller, S.G., Meinnel, T., Arnesen, T., Giglione, C., Hell, R., Wirtz, M. (2015) Proteome imprinting by N-terminal acetylation is a vital hormone-regulated switch during drought stress. Nat. Comm. 6:7640.doi: 10.1038/ncomms8640

Xu, F., Huang, Y., Li, L., Gannon, P., Linster, E., Huber, M., Kapos, Z., Bienvenut, W., Polevoda, B., Meinnel, T., Hell, R., Giglione, C., Zhang, Y., Wirtz, M., Chen, S. (2015) Two N-terminal acetyltransferases antagonistically control the stability of a Nod-like receptor in plant. Plant Cell, Vol. 27: 1547–1562, doi: 10.1105/tpc.15.00173

Yang, Y., 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: 1272-82, doi: 10.1111/mmi.13006

Dietz, K.-J., Hell, R. (2015) Thiol switches in redox regulation of chloroplasts: balancing redox state, metabolism and oxidative stress. Biol. Chem. DOI: 10.1515/hsz-2014-0281

Tavares, S., Wirtz, M., Beier, M., Bogs, J., Hell, R., Amâncio, S. (2015) Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants. Front. Plant Sci. 6:74, doi: 10.3389/fpls.2015.00074 

Speiser, A., Haberland, H., Watanabe, M., Wirtz, M., Dietz, K.-J., Saito, K., Hell, R. (2015) The significance of cysteine synthesis for acclimation to high light conditions. Front. Plant Sci. 5:775; doi: 10.3389/fpls.2014.00776

Birke, H., Hildebrandt, T.M., Wirtz, M., Hell, R. (2015) Sulfide detoxification in plant mitochondria. Meth. Enzymol. 555: 271-286, doi:10.1016/bs.mie.2014.11.027

2014

Forieri, I., Hell, R. (2014) Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic Interactions in Plants. In: Nutrient Use Efficiency in Plants: Approaches and Concepts. Hawkesford, M., Kopriva, S., De Kok, L.J. (eds.) Springer Publ., pp. 133 – 152; Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_5

Prioretti, L., Gontero, B., Hell, R., Giordano, M. (2014) Diversity and regulation of ATP sulfurylase in photosynthetic organisms. Front. Plant Physiol. 5, doi: 10.3389/fpls.2014.00597

Birke, H., De Kok, L.J., Wirtz, M., Hell, R. (2014) The role of compartment-specific cysteine synthesis for sulfur homeostasis during H2S exposure in Arabidopsis. Plant Cell Physiol., 56: 358-67. doi: 10.1093/pcp/pcu166

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; doi:10.3390/metabo4030629

Krüßel, L., Junemann, J., Wirtz, M., Birke, H., Thornton, J.D., Browning, L.W., Poschet, G., Hell, R., Balk, J., Braun, H.-P., Hildebrandt, T.M. (2014) The mitochondrial sulfur dioxygenase ETHE1 is required for amino acid catabolism. Plant Physiol. 165: 92 - 104

Cao, M.-J., Wang, Z., Q., Mao, J.-L., Speiser, A., Wirtz, M., Hell, R., Zhu, J.-K., Xiang, C.-B. (2014) Sulfate availability affects ABA levels and germination response to ABA and salt stress in Arabidopsis thaliana. Plant J. 77: 604-15

Lee, C.-P., Wirtz, M., Hell, R. (2014) Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana. Plant Cell Physiol. 55: 64–73

Albrecht, S.C., Sobotta,M.C., Bausewein, D., Aller, I., Hell, R., Dick, T.P., Meyer, A.J. (2014) Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes. J. Biomol. Screen. 19: 379 – 386

Khan, M.S., Hell, R. (2014) Applied Cell Biology of Sulfur and Selenium in Plants. In: Applied Plant Cell Biology: Cellular Tools and Approaches for Plant Biotechnology. Nick, P., Opatrný, Z., eds., Plant Cell Monographs, Springer, Heidelberg

 

 

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.
PDF 
Lee CP, Wirtz M, Hell R. (2013).
Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.
Plant Cell Physiol. x: y-z. 
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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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 
 

 

 

 

2012

Cao MJ, Wang Z, Wirtz M, Hell R, Oliver DJ, Xiang CB. (2012).
SULTR3;1 is a chloroplast-localized sulfate transporter in Arabidopsis thaliana.
Plant J. x: y - z. 
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.
Pubmed 
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 

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.
PDF 
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.
PDF 
Takahashi H, Kopriva S, Giordano M, Saito K, Hell R. (2011).
Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes.
Annu Rev Plant Biol. 62:157-84.
Abstract
Sulfur is required for growth of all organisms and is present in a wide variety of metabolites having distinctive biological functions. Sulfur is cycled in ecosystems in nature where conversion of sulfate to organic sulfur compounds is primarily dependent on sulfate uptake and reduction pathways in photosynthetic organisms and microorganisms. In vascular plant species, transport proteins and enzymes in this pathway are functionally diversified to have distinct biochemical properties in specific cellular and subcellular compartments. Recent findings indicate regulatory processes of sulfate transport and metabolism are tightly connected through several modes of transcriptional and posttranscriptional mechanisms. This review provides up-to-date knowledge in functions and regulations of sulfur assimilation in plants and algae, focusing on sulfate transport systems and metabolic pathways for sulfate reduction and synthesis of downstream metabolites with diverse biological functions.
Pubmed 
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

Maughan SC, Pasternak M, Cairns N, Kiddle G, Brach T, Jarvis R, Haas F, Nieuwland J, Lim B, Müller C, Salcedo-Sora E, Kruse C, Orsel M, Hell R, Miller AJ, Bray P, Foyer CH, Murray JA, Meyer AJ, Cobbett CS. (2010).
Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses.
Proc Natl Acad Sci U S A. 107:2331-6. 
Abstract
In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.
Pubmed 
Harada E, Kim JA, Meyer AJ, Hell R, Clemens S, Choi YE. (2010).
Expression profiling of tobacco leaf trichomes identifies genes for biotic and abiotic stresses.
Plant Cell Physiol. 51:1627-37. 
Abstract
Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcription-PCR (RT-PCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants.
Pubmed 
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

Brach T, Soyk S, Müller C, Hinz G, Hell R, Brandizzi F, Meyer AJ. (2009).
Non-invasive topology analysis of membrane proteins in the secretory pathway.
Plant J. 57:534-41. 
Abstract
We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for AtERD2 with six transmembrane domains.
Pubmed 
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 
Dalakouras A, Moser M, Zwiebel M, Krczal G, Hell R, Wassenegger M. (2009).
A hairpin RNA construct residing in an intron efficiently triggered RNA-directed DNA methylation in tobacco.
Plant J. 60:840-51. 
Abstract
So far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis- and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.
Pubmed 


2008

Schwarzländer M, Fricker MD, Müller C, Marty L, Brach T, Novak J, Sweetlove LJ, Hell R, Meyer AJ. (2008).
Confocal imaging of glutathione redox potential in living plant cells.
J Microsc. 231:299-316. 
Abstract
Reduction-oxidation-sensitive green fluorescent protein (roGFP1 and roGFP2) were expressed in different sub-cellular compartments of Arabidopsis and tobacco leaves to empirically determine their performance as ratiometric redox sensors for confocal imaging in planta. A lower redox-dependent change in fluorescence in combination with reduced excitation efficiency at 488 nm resulted in a significantly lower dynamic range of roGFP1 than for roGFP2. Nevertheless, when targeted to the cytosol and mitochondria of Arabidopsis leaves both roGFPs consistently indicated redox potentials of about -320 mV in the cytosol and -360 mV in the mitochondria after pH correction for the more alkaline matrix pH. Ratio measurements were consistent throughout the epidermal cell layer, but results might be attenuated deeper within the leaf tissue. Specific interaction of both roGFPs with glutaredoxin in vitro strongly suggests that in situ both variants preferentially act as sensors for the glutathione redox potential. roGFP2 targeted to plastids and peroxisomes in epidermal cells of tobacco leaves was slightly less reduced than in other plasmatic compartments, but still indicated a highly reduced glutathione pool. The only oxidizing compartment was the lumen of the endoplasmic reticulum, in which roGFP2 was almost completely oxidized. In all compartments tested, roGFP2 reversibly responded to perfusion with H(2)O(2) and DTT, further emphasizing that roGFP2 is a reliable probe for dynamic redox imaging in planta. Reliability of roGFP1 measurements might be obscured though in extended time courses as it was observed that intense irradiation of roGFP1 at 405 nm can lead to progressive photoisomerization and thus a redox-independent change of fluorescence excitation ratios.
Pubmed 
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

Meyer AJ, Brach T, Marty L, Kreye S, Rouhier N, Jacquot JP, Hell R. (2007).
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer.
Plant J. 52:973-86. 
Abstract
The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.
Pubmed 
Grzam A, Martin MN, Hell R, Meyer AJ. (2007).
gamma-Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis.
FEBS Lett. 581:3131-8. 
Abstract
The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.
Pubmed 
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 
Kruse C, Jost R, Lipschis M, Kopp B, Hartmann M, Hell R. (2007).
Sulfur-enhanced defence: effects of sulfur metabolism, nitrogen supply, and pathogen lifestyle.
Plant Biol 9:608-19. 
Abstract
Evidence from field experiments indicates differential roles of sulfur and nitrogen supply for plant resistance against pathogens. Dissection of these observations in defined pathosystems and controlled nutritional conditions indicates an activation of plant sulfur metabolism in several incompatible and compatible interactions. Contents of cysteine and glutathione as markers of primary sulfate assimilation and stress response show increases in ARABIDOPSIS THALIANA upon infection, coinciding with the synthesis of sulfur-containing defence compounds. Similar increases of thiols were observed with necrotrophic, biotrophic, and hemibiotrophic pathogens. Sulfate supply was found to be neutral or beneficial for tolerance against fungal but neutral for bacterial pathogens under IN VITRO conditions. According to various reports and own observations the effects of nitrogen supply appeared to be neutral or harmful, depending on the pathogen. The activation of sulfur metabolism was a consequence of activation of gene expression as revealed by macroarray analysis of an A. THALIANA/ALTERNARIA BRASSICICOLA pathosystem. This activation appeared to be largely independent from sufficient or optimal sulfate supply and from the established sulfate deficiency response. The data suggest that plant-pathogen interactions and sulfur metabolism are linked by jasmonic acid as signal.
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 
Grzam A, Tennstedt P, Clemens S, Hell R, Meyer AJ. (2006).
Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase.
FEBS Lett. 580:6384-90. 
Abstract
Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione-bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the gamma-glutamylcysteine-bimane intermediate (gamma-EC-B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine-bimane without intermediate. Only co-infiltration of monochlorobimane with Cd2+ and Cu2+ increased gamma-EC-B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null-mutants. In the absence of elevated heavy metal concentrations glutathione-conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate-limiting.
Pubmed 


2005

Wiedemuth K, Müller J, Kahlau A, Amme S, Mock HP, Grzam A, Hell R, Egle K, Beschow H, Humbeck K.(2005).
Successive maturation and senescence of individual leaves during barley whole plant ontogeny reveals temporal and spatial regulation of photosynthetic function in conjunction with C and N metabolism.
J Plant Physiol. 162:1226-36. 
Abstract
During ontogeny of barley plants (Hordeum vulgare, L. cv. Barke), a continuous developmental gradient of new leaves at the top and lower leaves undergoing senescence is maintained. In the course of senescence, specific recycling processes efficiently transfer valuable resources, e.g. nitrogen and carbon, to the growing young leaves and ears. In order to understand the temporal and spatial sequence of processes underlying this developmental program of leaf formation and degradation, changes in photosynthetic parameters, as well as C and N levels of all individual leaves were determined. During whole plant ontogeny, a strict sequential pattern of incorporation and degradation of C and N resources in the individual leaves, accompanied by a sequential loss of chlorophyll and photosynthetic function, was observed. In addition, protein levels of key enzymes of C and N anabolism AGPase (ADPglucose pyrophosphorylase) and GS (glutamine synthetase; plastidic isoform) also showed a strict pattern of sequential down-regulation in senescing leaves. Their decline preceded the breakdown of chlorophyll, total C and N levels and photosynthetic performance in the leaves. Quantitative real time PCR measurements revealed that the down-regulation of protein content of AGPase and GS correlated with a drastic decrease in their transcript levels. These data elucidated precise temporal and spatial regulation of C and N metabolism and allocation with photosynthetic function in the leaves during whole plant ontogeny of barley.
Pubmed 
Hell R, Leustek T. (2005).
Sulfur metabolism in plants and algae--a case study for an integrative scientific approach.
Photosynth Res. 86(3):297-8. 
Abstract
No abstract available
Pubmed 
Norici A, Hell R, Giordano M. (2005).
Sulfur and primary production in aquatic environments: an ecological perspective.
Photosynth Res. 86:409-17. 
Abstract
Sulfur is one of the critical elements in living matter, as it participates in several structural, metabolic and catalytic activities. Photosynthesis is an important process that entails the use of sulfur during both the light and carbon reactions. Nearly half of global photosynthetic carbon fixation is carried out by phytoplankton in the aquatic environment. Aquatic environments are very different from one another with respect to sulfur content: while in the oceans sulfate concentration is constantly high, freshwaters are characterized by daily and seasonal variations and by a wide range of sulfur concentration. The strategies that algal cells adopt for energy and resource allocation often reflect these differences. In the oceans, the amount and chemical form of sulfur has changed substantially during the course of the Earth's history; it is possible that sulfur availability played a role in the evolution of marine phytoplankton communities and it may continue to have appreciable effects on global biogeochemistry and ecology. Phytoplankton is also the main biogenic source of sulfur; sulfur can be released into the atmosphere by algal cells as dimethylsulfide, with possibly important repercussions on global climate. These and related matters are discussed in this review.
Pubmed 
Giordano M, Norici A, Hell R. (2005).
Sulfur and phytoplankton: acquisition, metabolism and impact on the environment.
New Phytol. 166:371-82. 
Abstract
Sulfur emission from marine phytoplankton has been recognized as an important factor for global climate and as an entry into the biogeochemical S cycle. Despite this significance, little is known about the cellular S metabolism in algae that forms the basis of this emission. Some biochemical and genetic evidence for regulation of S uptake and assimilation is available for the freshwater model alga Chlamydomonas. However, the marine environment is substantially different from most fresh waters, containing up to 50 times higher free sulfate concentrations and challenging the adaptive mechanisms of primary and secondary S metabolism in marine algae. This review intends to integrate ecological and physiological data to provide a comprehensive view of the role of S in the oceans.
Pubmed 
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 
Meyer AJ, Hell R.(2005).
Glutathione homeostasis and redox-regulation by sulfhydryl groups.
Photosynth Res. 86:435-57. 
Abstract
Continuous control of metabolism and developmental processes is a key feature of live cells. Cysteine thiol residues of proteins are both exceptionally useful in terms of structural and regulatory aspects, but at the same time exceptionally vulnerable to oxidation. Conserved cysteines thus are highly important for the function of metabolic enzymes and for signaling processes underlying responses to environmental factors. The underlying mechanism for the central role of thiol-mediated redox control in cellular metabolism is the ability of the cysteine-thiols to reversibly change their redox state followed by changes of structural, catalytic or regulatory functions. The cellular glutathione/glutathione disulfide redox buffer is present in cells at millimolar concentrations and forms one major basis of redox homeostasis by which protein thiols can maintain their redox state or oxidized protein thiols can be reverted to their reduced state. Besides acting as redox buffer, glutathione also acts as an electron donor for both scavenging of reactive oxygen, e.g. from photosynthesis and respiration, and metabolic reactions such as reduction of hydroperoxides and lipidperoxides or sulfate assimilation. The central role of glutathione is further emphasized by its involvement in signaling processes and the crosstalk of redox signaling processes with other means of signaling including protein glutathionylation and control of transcription factors. The present review aims at highlighting the key functions of glutathione in thiol-mediated redox control and its interplay with other protein-thiol-based redox systems.
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 
Douchkov D, Grycgka C, Stephan UW, Hell R, Baeumlein H. (2005).
Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco.
Plant Cell Environm. 28:365-74. 
Abstract
No abstract available
Pubmed 


2004

Kawashima CG, Berkowitz O, Hell R, Noji M, Saito K. (2004). Characterization and expression analysis of a serine acetyltransferase gene family involved in a key step of the sulfur assimilation pathway in Arabidopsis.
Plant Physiol. 137:220-30. 
Abstract
Ser acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetyl-Ser from L-Ser and acetyl-CoA, leading to synthesis of Cys. According to its position at the decisive junction of the pathways of sulfur assimilation and amino acid metabolism, SATases are subject to regulatory mechanisms to control the flux of Cys synthesis. In Arabidopsis (Arabidopsis thaliana) there are five genes encoding SATase-like proteins. Two isoforms, Serat3;1 and Serat3;2, were characterized with respect to their enzymatic properties, feedback inhibition by L-Cys, and subcellular localization. Functional identity of Serat3;1 and Serat3;2 was established by complementation of a SATase-deficient mutant of Escherichia coli. Cytosolic localization of Serat3;1 and Serat3;2 was confirmed by using fusion construct with the green fluorescent protein. Recombinant Serat3;1 was not inhibited by L-Cys, while Serat3;2 was a strongly feedback-inhibited isoform. Quantification of expression patterns indicated that Serat2;1 is the dominant form expressed in most tissues examined, followed by Serat1;1 and Serat2;2. Although Serat3;1 and Serat3;2 were expressed weakly in most tissues, Serat3;2 expression was significantly induced under sulfur deficiency and cadmium stress as well as during generative developmental stages, implying that Serat3;1 and Serat3;2 have specific roles when plants are subjected to distinct conditions. Transgenic Arabidopsis plants expressing the green fluorescent protein under the control of the five promoters indicated that, in all Serat genes, the expression was predominantly localized in the vascular system, notably in the phloem. These results demonstrate that Arabidopsis employs a complex array of compartment-specific SATase isoforms with distinct enzymatic properties and expression patterns to ensure the provision of Cys in response to developmental and environmental changes.
Pubmed 
Bauer P, Thiel T, Klatte M, Bereczky Z, Brumbarova T, Hell R, Grosse I. (2004).
Analysis of sequence, map position, and gene expression reveals conserved essential genes for iron uptake in Arabidopsis and tomato.
Plant Physiol. 136:4169-83. 
Abstract
Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.
Pubmed 
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

Rutten T, Krüger C, Melzer M, Stephan UW, Hell R. (2003).
Discovery of an extended bundle sheath in Ricinus communis L. and its role as a temporal storage compartment for the iron chelator nicotianamine.
Planta. 217:400-6. 
Abstract
The extended bundle sheath (EBS) is a specialized layer of cells that enhances the lateral transport of photoassimilates within the leaf. This little-known tissue is often considered to be legume-specific. We identified an EBS in cotyledons and leaves of the non-legume Ricinus communis L. By means of cytological and immunological studies and using the localization of the iron-chelator nicotianamine as an established indicator for mass transport, we confirmed its role as a transport tissue and a temporal sink. Observations on cotyledons of Ricinus seedlings further proved that the EBS carries out these tasks from a very early stage of development onwards. This is the first time that information has been obtained on the physiological role of an EBS in a non-legume. Our results support the idea of its widespread occurrence among higher plants.
Pubmed 
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 
Schmiedeberg L, Krueger C, Stephan UW, Baeumlein H, Hell R. (2003).
Synthesis and proof-of-function of a [14C]-labelled form of the plant iron chelator nicotianamine using recombinant nicotianamine synthase from barley.
Physiol Plant.118:430-8. 
Abstract
No abstract available
Pubmed 


2002

Hell R, Stephan UW. (2002).
Iron uptake, trafficking and homeostasis in plants.
Planta. 216:541-51. 
Abstract
Iron is an essential micronutrient with numerous cellular functions, and its deficiency represents one of the most serious problems in human nutrition worldwide. Plants have two major problems with iron as a free ion: its insolubility and its toxicity. To ensure iron acquisition from soil and to avoid iron excess in the cells, uptake and homeostasis are tightly controlled. Plants meet the extreme insolubility of oxidized iron at neutral pH values by deficiency-inducible chelation and reduction systems at the root surface that facilitate uptake. Inside the cells the generation of highly toxic hydroxyl radicals by iron redox changes is avoided by intricate chelation mechanisms. Organic acids, most notably nicotianamine, and specialized proteins bind iron before it can be inserted into target molecules for biological function. Uptake and trafficking of iron throughout the plant is therefore a highly integrated process of membrane transport and reduction, trafficking between chelator species, whole-plant allocation and genetic regulation. The improvement of crop plants with respect to iron efficiency on iron-limiting soils and to iron fortification for human nutrition has been initiated by breeding and biotechnology. These efforts have to consider molecular and physiological evidence to overcome the inherent barriers and problems of iron metabolism.
Pubmed 
Kruger C, Berkowitz O, Stephan UW, Hell R. (2002).
A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L.
J Biol Chem. 277:25062-9. 
Abstract
The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.
Pubmed 
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

Hell R, Hillebrand H. (2001).
Plant concepts for mineral acquisition and allocation.
Curr Opin Biotechnol. 12:161-8. 
Abstract
Plant biotechnology is expected to make a major contribution to the steady increase of crop production in the near future. The improvement of mineral assimilation has to meet the challenges of reducing fertilizer application in developed countries, preserving the environment, enabling sustainable agriculture management and generating low-input crops with increased performance in areas where soil infertility limits productivity. Natural genetic resources and engineered plants will help to achieve the implementation of traits for improved mineral assimilation.
Pubmed 
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/ Prof. Dr. Rüdiger Hell _e