Foreword (I): Sulfur metabolism - Yolande Surdin-Kerjan
Foreword (II): Sulfur nutrition and sulfur assimilation of higher plants -
The plant sulfate transporter family: Specialized functions and integration with whole plant nutrition -
Malcolm J. Hawkesford, Peter Buchner,Laura Hopkins and Jonathan R. Howarth
Molecular and metabolic regulation of sulfur assimilation: initial approach by the post-genomics strategy -
Metabolic regulation of cysteine synthesis and sulfur assimilation –
Regulation of sulfate uptake: from molecular bases to whole plant integration - Lessons from research on the regulation of nitrate uptake -
Bruno Touraine, Timothy J. Tranbarger and Patricia Nazoa
Interactions between sulfur, nitrogen and carbon metabolism -
Christian Brunold, Peter Von Ballmoos, Holger Hesse, David Fell and Stanislav Kopriva
S-Methylmethionine and the interface between sulfur and one-carbon metabolism -
Sanja Roje and Andrew D. Hanson
Genetic and molecular analysis of phytochelatin biosynthesis, regulation and function -
Christopher S. Cobbett
Physiological and environmental significance of selenium -
Danika L. Leduc and Norman Terry
Heavy metal regulation of cysteine biosynthesis and sulfur metabolism related to stress in Arabidopsis thaliana trichomes - Cecilia Gotor, José R. Domínguez-Solís, Gloria Gutiérrez-Alcalá, M. Carmen López-Martín, Leticia Calo and Luis C. Romero
Roles of glutathione and glutathione-related enzymes in remediation of polluted soils by transgenic poplars -
Tamás Kömives, Gabor Gullner and Heinz Rennenberg
Sulfur nutrition of deciduous trees at different environmental growth conditions -
Impact of volatile sulfur compounds on wine quality -
Importance of sulfur for the quality of breadmaking wheat and malting barley -
Fang-Jie Zhao and Steve P. McGrath
Contributed papers: (in alphabetical order)
Effect of N and S fertilization on the glucosinolate content of leaves and roots of broccoli seedlings -
Alfredo Aires, Rosa Carvalho, Eduardo Rosa and Fernanda Maria Pereira
Screening of mutants accumulating high levels of anions and thiols from activation-tagged lines of Arabidopsis thaliana - Motoko Awazuhara, Akiko Hayase, Masatomo Kobayashi, Masaaki Noji, Mami Yamazaki, Motoaki Seki, Kazuo Shinozaki and Kazuki Saito
Comparison of sulfur deficiency indicators in winter oilseed rape -
Ursula Balestra, Mechteld Blake-Kalff, J.-C. Lavanchy, A. Keiser, H.-R. Pfeifer and D. Pellet
Further investigation on the role of the prolonged linker sequence in plant sulfur transferases -
Michael Bauer and Jutta Papenbrock
Application of biacore technology for the analysis of the protein interactions within the cysteine synthase complex -
Oliver Berkowitz, Markus Wirtz, Alexander Wolf, Jürgen Kuhlmann and Rüdiger Hell
Influence of sulfur availability on non-protein thiols contents in transgenic tobacco lines producing bacterial serine acetyltransferase -
Anna Blaszczyk and Agnieszka Sirko
Influence of the sulfur supply on the (iso)alliin content in leaves and bulbs of Allium cepa and Allium sativum -
Elke Bloem, Silvia Haneklaus and Ewald Schnug
Model based prognosis of sulfur deficiency -
Elke Bloem, Silvia Haneklaus and Ewald Schnug
Glutathione transporter homologues from Arabidopsis thaliana -
Olivier Cagnac, Andree Bourbouloux and Serge Delrot
Sulfur status and acquisition in response to different sulfate concentrations and time exposure in maize -
David Carden, Silvia Quaggiotti and Mario Malagoli
Nitrogen and sulfur requirement of Brassica oleracea L. cultivars -
Ana Castro, Ineke Stulen and Luit J. De Kok
Glutathione content and status of in vitro plantlets transferred to ex vitro: demand for growth or response to oxidative stress? -
Luísa De Carvalho and Sara Amâncio
Sulfur dynamics in fallow soil and in the rhizosphere of field-grown rape and barley -
Odile Dedourge, Phuy-Chhoy Vong, Françoise Lasserre-Joulin, Emile Benizri and Armand Guckert
O-acetylserine (thiol)lyase confers tolerance to heavy metals -
R. Domínguez-Solís, M. Carmen López-Martín, M. Dolores Ynsa, Francisco J. Ager, Luis C. Romero and Cecilia Gotor
Impact of atmospheric H2S on sulfur and nitrogen metabolism in Allium species and cultivars -
Mark Durenkamp and Luit J. De Kok
Identification and characterization of Arabidopsis thaliana mutants resistant to selenate -
Elie El Kassis, Nicole Cathala, Pierre Fourcroy, Pierre Berthomieu, Abderrahmane Tagmount, Norman Terry and Jean-Claude
The effect of catch crops on sulfate leaching and availability of sulfur in the succeeding crop -
Jørgen Eriksen and Kristian Thorup-Kristensen
Characterization and differential expression of two sulfate transporters in Arabidopsis thaliana -
Pierre Fourcroy, Elie El Kassis, Elisa Dalla Pria, Nicole Cathala and Jean-Claude Davidian
Thiols in acorns and feeding mites collected at sites with naturally elevated atmospheric sulfur concentrations -
Dieter Grill, Michael Tausz, B. Strnad, Astrid Wonisch, Maria Müller and Antonio Raschi
Role of ATP sulfurylase in the regulation of the sulfate assimilation pathway in plants -
Yves Hatzfeld, Nicole Cathala, Dao-Yao He and Jean-Claude Davidian
Investigation of the Fe-S cluster biosynthesis in higher plants -
Nicole Hausmann, Holger Eubel and Jutta Papenbrock
Transcriptome analysis of sulfur-starved Arabidopsis by DNA array: OAS is a positive regulator of gene expression under sulfur deficiency -
Masami Yokota Hirai, Toru Fujiwara, Motoko Awazuhara, Tomoko Kimura, Masaaki Noji and Kazuki Saito
Transcript profiling of sulfur depletion in Arabidopsis thaliana -
Rainer Höfgen, Victoria Nikiforova, Jens Freitag, Stefan Kempa, Kerstin Riedel and Holger Hesse
Tissue and cell specific localisation of a sulfate transporter in maize -
Laura Hopkins and Malcolm J. Hawkesford
The role of elemental sulfur in tomato as a phytoalexin response to verticillium infection -
Jonathan R. Howarth, Jane S. Williams, Richard M. Cooper and Malcolm J. Hawkesford
Selection of methionine-enriched soybean seeds -
John Imsande and M. Paul Scott
COR - an Arabidopsis thaliana protein with cystine lyase activity -
Patrik Jones and Kazuki Saito
New approaches to study "sulfur-induced resistance" against fungal pathogens in Arabidopsis thaliana -
Ricarda Jost, Paul Scholze and Rüdiger Hell
Temporal and spatial expression analysis of serine acetyltransferase isoforms in Arabidopsis thaliana -
Cintia G. Kawashima, Masaaki Noji and Kazuki Saito
The effect of lead on sulfoquinovosyl diacylglycerol content in leaves and roots of wheat seedlings -
Oksana Kosyk, Alexander Okanenko and Nataliya Taran
Grass sulfur status and non-protein nitrogen accumulation: a preview in the southeastern part of Belgium -
Richard Lambert, Michaël Mathot, Bernard Toussaint and Alain Peeters
Comparison of oilseed rape and barley rhizosphere microbial communities involved in sulfur immobilization - preliminary results -
Françoise Lasserre-Joulin, Romain Boulan, Emile Benizri, Phuy-Chhoy Vong and Armand Guckert
Identification and characterization of the AtNFS2 gene from Arabidopsis thaliana encoding a nifs-like plastidial cysteine desulfurase -
SebastienLeon, Brigitte Touraine, Jean-François Briat and Stephane Lobreaux
Analysis of transgenic tobacco lines expressing bacterial CYSK gene encoding O-acetylserine (thiol)lyase A -
Frantz Liszewska and Agnieszka Sirko
Effects of nitrogen availability on sulfate uptake in common reed -
Mario Malagoli, Claudia Bragato and Anna-Rita Trentin
Localization of g-glutamyl-transferase activity in plant tissue -
Antonio Masi, Tiziana Destro and Massimo Ferretti
Effects of nitrogen and sulfur fertilization on grass yield and quality in Belgium -
M. Mathot, R. Lambert, J. Mertens, B. Toussaint and A. Peeters
Genomic and biochemical studies of sulfur assimilation in onion -
John McCallum, Meeghan Pither-Joyce, Martin Shaw, Anya Lambert and Michael T. McManus
Effect of mineral sulfur and organic fertilizers on yield and sulfur uptake of grass on Belgian sandy soils -
J. Mertens, G. Verlinden and M.Geypens
Arabidopsis sulfur transferases: investigation on their role in the organism -
Tanja Meyer, Meike Burow, Michael Bauer and Jutta Papenbrock
Subcellular distribution of glutathione - a high resolution immunogold analysis in leaves of pumpkin (Cucurbita pepo L.) -
Maria Müller, Bernd Zechmann, Michael Tausz and Günther Zellnig
Cluster analysis of gene responses to sulfur depletion in Arabidopsis thaliana-
Victoria Nikiforova, Carsten Daub, Jens Freitag, Stefan Kempa, Kerstin Riedel, Holger Hesse and Rainer Höfgen
Altered expression of serine acetyltransferase gene in transgenic Arabidopsis resulted in modulated production of cysteine and glutathione-
Masaaki Noji, Fumiko Saito, Tomoko Ochiai, Yumiko Shirano, Hiroaki Hayashi, Daisuke Shibata, Tomohiko Kato, Satoshi Tabata and Kazuki Saito
Transgenic Arabidopsis thaliana expressing GFP in response to sulfur nutrition -
Naoko Ohkama, Yoshitaka Sogawa, Derek B. Goto, Kentaro Takei, Hitoshi Sakakibara, Nakako Shibagaki, Hiroaki Hayashi, Tadakatsu Yoneyama, Satoshi Naito and Toru Fujiwara
Healthiness of winter oilseed rape fertilized with nitrogen and sulfur -
Anna Podlesna, Malgorzata Jedryczka and Elzbieta Lewartowska
Glutathione transport in pedunculate oak roots and its comparison with sulfate and glutamine transport -
Heinz Rennenberg and Stefan Seegmüller
Successful engineering of methionine metabolism in potato -
Kerstin Riedel, Michaela Zeh, Anna Paola Casazza, Rainer Höfgen and Holger Hesse
Effects of sulfur nutrition on carbon metabolism and nitrogen assimilation in Chlorella sorokiniana -
Carmelo Rigano, Simona Carfagna, Vittoria Di Martino Rigano, Vincenza Vona, Sergio Esposito and Graziella Massaro
Explaining sulfur and nitrogen interactions in the growth of cropped hybrid ryegrass -
Maria Da Graça Serrão, Michael Tausz, Maria João Neves, John Keith Syers and Fernando Pires
Selenate resistant mutants of Arabidopsis thaliana identifies a region in sulfate transporter gene required for efficient transport of sulfate in roots -
Nakako Shibagaki, Alan Rose, Toru Fujiwara, John P. Davies and Arthur R. Grossman
Sulfur uptake by rye grass as affected by different sulfur sources contained in calcium nitrate -
Bal Ram Singh and Leif Ruud
Jungermanniidae species respond to cadmium in a different manner to other bryophytes -
Kristin Sutter, Sieglinde Menge, Herbert Tintemann, Angelika Schierhorn and Gerd-Joachim Krauss
Sulfur transport and assimilation in developing embryos of chickpea (Cicer arietinum) -
Linda M. Tabe, Ingrid Venables, Anita Grootemaat and David Lewis
T-DNA insertion mutagenesis of sulfate transporters in Arabidopsis –
Hideki Takahashi, Akiko Watanabe-Takahashi and Tomoyuki Yamaya
Modification of sulfur metabolism in spruce trees by H2S studied by radiolabelled sulfate uptake -
Michael Tausz, Astrid Wonisch, Wilfried Weidner, Luit J. De Kok and Dieter Grill
Biosynthesis of cysteine and glutamate in Chlamydomonas reinhardtii: effect of nitrate or sulfate starvation and cadmium stress -
José M. Vega, Antonio Benitez-Burraco, Javier Vigara and Carlos Vilchez
Mobilization of 35S in rhizosphere soil of rape and barley: relationship between root-35S uptake and soil arylsulfatase activity -
Phuy-Chhoy Vong, Françoise Lasserre-Joulin and Armand Guckert
Wool quality and sulfur supply -
Shiping Wang, Yanfen Wang, Zuozhong Chen, Ewald Schnug and Silvia Haneklaus
Comparative biochemical characteri-zation of OAS-TL isoforms from Arabidopsis thaliana -
Markus Wirtz and Rüdiger Hell
Effects of SO2 exposure on sulfur distribution in curly kale (Brassica oleracea L.) Investigated by 35S-labelled nutrient solution -
Astrid Wonisch, Wilfried Weidner, Michael Tausz, Sue Westerman, Luit J. De Kok and Dieter Grill
Interaction between atmospheric sulfur dioxide deposition and pedospheric sulfate nutrition in chinese cabbage -
Liping Yang, Ineke Stulen and Luit J. De Kok
Characterization of two functional high-affinity sulfate transporters for uptake of sulfate in Arabidopsis roots -
Naoko Yoshimoto, Hideki Takahashi, Frank W. Smith, Tomoyuki Yamaya and Kazuki Saito
Influence of sulfur nutrition on sugar beet resistance to aphids -
Eva Zelená and Frantisek Zeleny
Influence of sulfur on growth of radish -
Frantisek Zeleny and Eva Zelená
Index of authors
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This book contains the invited and contributed papers of the 5th Workshop on Sulfur Transport and Assimilation in Plants, a joined European Commission (COST Action 829) and OECD meeting hosted at the Ecole Nationale Supérieure Agronomique in
Montpellier (France) from April 11 to 14, 2002. The meeting was co-organized by the ENSA-Montpellier (France), the University of Graz (Austria), the University of Groningen (The Netherlands), Rothamsted Research, (United Kingdom), Institute of Plant Nutrition and Soil Science, Braunschweig (Germany), the Agricultural Biotechnical Center of Gödöllö (Hungary), Albert-Ludwigs-University Freiburg (Germany) and the University of Chiba (Japan).
We are very pleased to dedicate this book to Prof. Dr. Christian Brunold, University of Bern, Switzerland. His views and research have significantly contributed to advanced understanding of the physiological, biochemical and molecular regulation of the sulfur metabolism pathway and its interactions with nitrogen metabolism. We also dedicate this book to Dr. Yolande Surdin-Kerjan, CNRS, Gif-sur-Yvette, France, whose outstanding studies on genetics and molecular biology have contributed
to the identification of most of the genes involved in the regulation of the sulfur metabolism pathway of the yeast Saccharomyces cerevisiae.
Luit J. De Kok
Malcolm J. Hawkesford
Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France
I entered the “Ecole Nationale Supérieure de Chimie de Paris” with the intent of becoming a chemical engineer and then working in industry. After graduation I was confronted with two facts: chemical industry was hostile to the presence of women and anyhow, I was not good enough at it to consider the life with chemistry. I thus went to visit Dr. Françoise Labeyrie who was a friend of my family and whose laboratory was devoted to the study of cytochromes. It took her only four hours to persuade me that Biology was the research field to join. She introduced me to Dr. Huguette de Robichon-Szulmajster who just created her research group to study the metabolism of amino acids deriving from aspartate in Saccharomyces cerevisiae. Huguette wanted to use S. cerevisiae as a model organism in a world then almost completely devoted to E. coli. I was naive enough not to realize that, so this point did not bother me, but I must say that, during my thesis work, I moderately appreciated the remarks that I heard from time to time (“yeast ?, why not rhinoceros…”). So, in September 1962, I got a position in CNRS, and joined Huguette's laboratory.
It was only in 1969, after obtaining my Ph.D. that I switched to methionine metabolism the study of which had been initiated by Hélène Chérest (who still works with me). In 1974 Huguette died from a cancer without knowing how good had been her intuition to use yeast as a model organism. I had to take over and during the next 10 years, we characterized many genes involved in the sulfur amino acids metabolism. More particularly we isolated the first yeast strains impaired in sulfate transport. During these years also, I met Dominique Thomas who came in the laboratory as a student and stayed. I thank him for staying, for being so enthusiastic about the study of the regulation of transcription of the sulfur amino acids metabolism, which he turned into a very sophisticated model and for sharing my interest in metabolism.
In 1992, I met Jean-Claude Davidian. He phoned me to get our sulfate transporter mutants, wanting to clone the plant sulfate transporter genes. Unfortunately, I had lost them. He was disappointed but talked me into trying to isolate such mutants again. He thus spent several months in my laboratory and, together with Hélène Chérest, successfully characterized and cloned the genes encoding the two yeast sulfate permeases. Meanwhile, Smith, Hawkesford, Prosser and Clarkson published the isolation of a yeast mutant devoid of sulfate transport. I still think they were paper, describing the isolation and characterization of the two sulfate permeases from S. cerevisiae, was published after theirs.
This is how I stepped into the world of plants. At the Montpellier meeting, it was a great pleasure for me to realize that our yeast mutants have been successfully used to clone several plant genes.
Sulfur amino acids metabolism in Saccharomyces cerevisiae
The biosynthetic pathway
Sulfur is an essential nutrient for all microorganisms. Extensive growth data has been accumulated showing that S. cerevisiae possesses various enzymatic systems that allow it to metabolize almost any sulfur source. In contrast to many other microorganisms, yeast is able to use various organic sulfur compounds, i.e. cysteine, methionine, homocysteine, glutathione, and S-adenosylmethionine (AdoMet) as a sole sulfur source. This is due to the organization of the sulfur pathway in yeast which allows the conversion of the main sulfur metabolites into the others. This pathway has been almost completely deciphered by my research group, using first biochemistry and classic genetics and later, the powerful reverse genetics methods. Finally, our work has also been greatly helped by the completion of the sequence determination of the yeast genome in 1996. To date, we have characterized more than 15 genes encoding enzymes of the sulfur amino acids pathway (Fig. 1).
In addition, we have shown that some mutants requiring methionine for growth bear mutations in genes encoding enzymes not directly related to the sulfur amino acids metabolism. These genes are MET19, MET22 and SOD1, encoding glucose-6- phosphate dehydrogenase, 3',(2'),5'-bisphosphate nucleotidase and one superoxide dismutase, respectively, as well as the less well characterized MET18 and MET27 genes. It is still unclear why mutations within these genes result in methionine auxotrophy, but it suggests that some functions of sulfur amino acids remain to be uncovered (Thomas and Surdin-Kerjan 1997). Recently, we have also characterized the FOL3 and the MET7 genes encoding the dihydrofolate synthetase and the folylpolyglutamate synthetase respectively, both involved in the addition of glutamate tails to folate coenzymes (Cherest et al. 2000). The fol3 and met7 mutants have been used recently to characterize the corresponding plant genes (Ravanel et al. 2001).
Recycling of the products of the catabolism of AdoMet
Methionine is not only involved in protein synthesis but is also an essential determinant of the one carbon metabolism. Indeed, under its activated form, Sadenosylmethionine (AdoMet), it is the methyl donor in numerous transmethylation reactions of nucleic acids, proteins or lipids. Further, AdoMet serves as a precursor for the biosynthesis of polyamines and is one of the substrates used in a number of reactions, including vitamin biosyntheses and nucleotide modifications.
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Fig. 1. Metabolism
of sulfur amino-acids in Saccharomyces cerevisiae. APS, adenosine 5'-
phosphosulfate; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; MTHFR, methylene tetrahydrofolate
reductase; CH3THF, methyl-tetrahydrofolate; CH2THF, methylene tetrahydrofolate; MTA,
methylthioadenosine; MTR, methylthioribose-1-phosphate; dSAM, decarboxylated AdoMet.
Given such ubiquitous functions, the equilibrium between methionine and AdoMet is thus expected to be of crucial importance for the overall cellular homeostasis. In eucaryotic cells, the methionine/AdoMet ratio was thought to be largely controlled through two recycling pathways that act on the products of AdoMet catabolism. The first one, called the methyl cycle, allows the conversion of Sadenosylhomocysteine, the by-product of all transmethylation reactions, into homocysteine which is next remethylated into methionine by the methionine synthase. The second one comprises a set of complex reactions that allow the direct synthesis of methionine from 5'-methylthioadenosine (MTA), a compound formed mainly during polyamine biosynthesis. In this pathway (called the MTA cycle), the ribose moiety of the adenosyl group gives rise to the four carbon skeleton of methionine while conserving the methylthiol group (Fig. 1).
Using molecular genetics, we have been able to prove that the methyl cycle as well as the MTA cycle are both active in S. cerevisiae. In addition, more recently, we have uncovered a pathway allowing the utilisation of S-methylmethionine by yeast, and we have shown that AdoMet can be used to directly methylate homocysteine, yielding methionine in what seems to be a futile cycle. However, this cycle is fully active when AdoMet is the sole sulfur source present in the growth medium (Thomas et al. 2000).
Transport of sulfur compounds
Transport systems allow yeast cells to extract virtually any sulfur compounds from their environment. Using specifically designed genetic screens, we have isolated and characterized: (i) the genes encoding the two sulfate transporters, Sul1p and Sul2p (Cherest et al. 1997). As I mentioned, the SUL1 gene was isolated first by Smith et al. (1995); (ii) the two methionine transporters, Mup1p and Mup3p (Isnard et al. 1996); (iii) the AdoMet transporter, Sam3p (Rouillon et al. 1999); and (iv) the Smethylmethionine transporter, Mmp1p (Rouillon et al. 1999).
In addition to these highly specific transport systems, biochemical uptake assays have revealed the presence in yeast of less specific transport systems for most of the sulfur compounds.
Regulation of sulfur amino-acids biosynthesis
The first observations about the MET regulatory system were made in the 1970's. We showed that when wild type S. cerevisiae cells are grown in the presence of a high concentration of methionine (1 mM), a decrease of the synthesis of all the enzymes implicated in methionine biosynthesis is measured. Later, we were able to show that this negative regulation is acting at the transcription level and that the signal was AdoMet. Then, we identified several cis-acting regulatory sequences found upstream of the MET genes and demonstrated that the negative regulation resulted from the non-activation of the transcription of the genes (Thomas et al. 1989).
During the 1990s, we isolated most of the genes encoding the factors responsible for the transcriptional regulation the MET genes, i.e. Met4p, Met28p, Met30p, Met31p and Met32p. Meanwhile, Cbf1p, a protein that functions at both centromeres and MET gene promoters was characterized in several laboratories and its precise role in MET gene regulation was deciphered in my laboratory. All these factors were identified either by specific genetic screens or through specific molecular assays, such as one hybrid experiments.
The identification of so many factors was suggestive of an unanticipated complexity of the molecular mechanisms underlying the regulation of the MET gene network. We have been able to decipher the precise function of all these factors. Briefly, a unique transcriptional activator, Met4p, is recruited to the MET promoters trough the assembly of different large multiprotein complexes (Cbf1p-Met28pMet4p and Met4p-Met28p-Met31p (Met32p)) (Kuras et al. 1996; Kuras et al. 1997; Blaiseau and Thomas 1998) Indeed, the binding of Met4p to DNA varies from one gene to another. Once tethered to DNA, Met4p activates the transcription of the downstream gene owing to an unique acidic activation domain. When the cells are exposed to a high methionine concentration, Met4p is inactivated through the activity of the SCFMet30 ubiquitin ligase and MET gene transcription is turned off. SCF ubiquitin ligases are multi-protein complexes, that use substrate-specific adapter subunits termed F-box proteins (such as Met30p) to recruit substrates for ubiquitylation by a core apparatus, which is composed of the scaffold protein Cdc53/cullin, the RING finger protein Rbx1, the adapter protein Skp1, and the E2 enzyme Cdc34. Surprisingly, the consequence of Met4 ubiquitylation differs according to the growth conditions. In cells grown in minimal medium, Met4 ubiquitylation triggers its degradation by the 26S proteasome (Rouillon et al. 2000). In contrast, in rich medium, ubiquitylated Met4p is stable but unable to bind to the MET promoters while ubiquitylated Met4p is still capable of activating the AdoMet biosynthesis genes. Thus, ubiquitylation not only regulates the MET gene network by distinct degradation-dependent and -independent mechanisms, but also controls the differential recruitment of Met4p, thereby diversifying its activation specificity (Kuras et al. 2002).
I am very grateful to the editors and more particularly to Jean-Claude Davidian for partly dedicating this volume to me. And I am happy to share this honour with Dr. Brunold whom I have learned to know during these last three sulfur meetings and whose great knowledge of plant sulfur metabolism I have come to appreciate. This honour is a great reward and well compensates me for the hard work during all these years. I think that I have lived during a fantastic period of time during which, starting with a partially known biosynthetic pathway, we could end by formulating a recognized model for the regulation of transcription. This was greatly helped by our knowledge of the metabolism itself. With the modern techniques that can be now used for plant metabolism, I hope that the young scientists will live rewarding moments as I did.
I thank my friend Professor Pete Magee for editing this manuscript and for being always present to help me when needed, ever since we met in Huguette's lab in 1965.
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SULFUR NUTRITION AND SULFUR ASSIMILATION OF HIGHER PLANTS
IPS, University of Berne, Altenbergrain 21, 3013 Bern, Switzerland
H2S as a starting point
I wish to thank the organizers of the 5th International Workshop on Sulfur Nutrition and Sulfur Assimilation in Higher Plants for their kind dedication of this distinguished volume containing many important contributions to the advancement of our knowledge of sulfur metabolism to Yolande Surdin-Kerjan and myself.
Sulfur metabolism was the main field of my scientific work since I started my Ph.D. thesis in 1969. At that time, my adviser, Prof. K.H. Erismann, suggested to find out, if higher plants could use electrons from H2S instead of H2O for producing reducing equivalents in photosynthesis. Since I was more interested in metabolism than in electron transport, I proposed to use H2S as sulfur source and to analyze, if it had a regulatory effect on sulfate assimilation. At that time we knew that Spirodela oligorrhiza (Ferguson 1969), but also Lemna minor, the organism preferentially analyzed in Erismann's laboratory, used NH4 + rather than NO3 - as a nitrogen source, when both ions were available in the nutrient solution. With this knowledge in mind, the obvious working hypothesis was that H2S rather than SO4 2- would be used as a sulfur source, when both were available. L. minor turned out to be cooperative, did exactly what we expected, and thus made it possible to finish the thesis in 1972. The results obtained can be summarized as follows: In L. minor gassed with 6 ppm H2S, uptake and assimilation of sulfate were almost completely inhibited. H2S taken up was partly directly used for cysteine formation, thus increasing the cysteine content (Brunold and Erismann 1975) and partly oxidized, which increased the sulfate content. The work with H2S treated L. minor had several very positive consequences, since (i) I was offered a position at the Institute of Plant Physiology at the University of Berne, Switzerland, and could start an academic career, (ii) I got a grant from the Swiss National Science Foundation (SNF) for working in the laboratory of Jerry Schiff at Brandeis University, which led to an inseparable tie with the field of sulfur metabolism, and to my first publication in an international plant journal, (Brunold and Schiff 1976), (iii) I got into contact with Ineke Stulen and Luit de was contacted by Ahlert Schmidt for a successful collaboration (Brunold and Schmidt 1976), and (v) I met Dr. Bergmann, University of Cologne, Germany, who brought me into contact with Heinz Rennenberg, with whom I collaborated till the end of my scientific career (Rennenberg and Brunold 1994; Hesse et al. 2003). Looking back, I consider it a great privilege that Dr. Erismann allowed me to work in sulfur metabolism for my Ph.D. thesis.
Answers and questions
After studying the regulation and the localization of sulfate assimilation for many years, I now make the experience many scientists made before me: There are more open questions, than when I started. This situation can be well exemplified using adenosine 5'-phosphosulfate (APS) reductase (APR, Setya et al. 1996; Suter et al. 2000), formerly named APS sulfotransferase (Schmidt 1972), my favored enzyme:
1. Like sulfate transporters (Takahashi et al. 2000; Vidmar et al. 2000), APR is especially sensitive to a lack (Brunold et al. 2002) or a surplus of reduced sulfur (Brunold and Schmidt 1976; Westerman et al. 2001; Vauclare et al. 2002). There are several indications (Takahashi et al. 2000; Vidmar et al. 2000; Vauclare et al. 2002) that GSH plays a role in signaling the sulfur status of plants, however, the genetic sequences, the genes and the mechanisms involved, which lead to induction or repression of APR during lack and surplus of sulfur, respectively, are not known.
2. The assimilation of sulfate and nitrate is regulated reciprocally in a coordinate manner (Brunold et al. 2002). O-acetyl-L-serine (OAS) seems to play a role in this coordination (Neuenschwander et al. 1991; Harms et al. 2000), but the mechanisms involved are not known. This is in contrast to the situation in bacteria, where regulatory mechanisms involving N-acetyl-L-serine (NAS), an isomer of OAS, have been elucidated in great detail (Kredich 2000), which can give indications for analyzing plant systems. APR may be the enzyme of choice for this analysis, because it is very sensitive to regulatory signals and is easy and fast to measure.
3. APR is regulated by sugars (Kopriva et al. 1999) and several mechanisms, which might be involved, have been described (Hesse et al. 2003). Regulation by sugars is also important in other systems, therefore, checking proposed mechanisms (Smeekens 2000; Hesse et al. 2003) and possibly new ideas using APR as an indicator of the sugar status may result in contributions of general relevance in the field of plant sugar sensing.
4. Calculation of the flux control coefficient of APR in A. thaliana root cultures indicated that APR had a high flux control coefficient, but was not rate limiting (Vauclare et al. 2002). The question is, if corresponding calculations in other systems, especially in green tissues, result in similar values for APR and in addition give values for the flux control coefficients of other enzymes of sulfate assimilation. Such a mathematical analysis would greatly help to understand the enzymatic regulation of this pathway.
5. In maize leaves, APR is localized exclusively in bundle sheath cells, and cysteine formed there is transported into the mesophyll cells, the predominant cell type for GSH synthesis (Burgener et al. 1998). Several ideas for explaining this special localization have been discussed, but an unequivocal physiological explanation is still missing. The question is complicated by the fact that the localization of sulfate assimilation in bundle sheath cells as detected in some C4 grasses is neither a prerequisite nor a consequence of C4 photosynthesis, since in dicot C4 Flaveria species, APR mRNA and protein were present at comparable levels in both types of cells (Koprivova et al. 2001).
6. It seems clear now that higher plants use APS as a sulfonyl donor in sulfate reduction, bacteria either use APS or adenosine 3'-phosphate 5'-phosphosulfate (PAPS), and in the moss Physcomitrella patens both APS and PAPS dependent sulfate assimilations coexist (Koprivova et al. 2002). The physiological, ecological and evolutionary causes for these different choices of sulfonyl donors are not known.
Scientists, Problems and Money
The interests of the scientists working in a certain field are changing with time, leaving some questions unanswered, focusing on others and possibly, but not necessarily, coming back to the old ones after some time for testing new ideas and new methods. It is therefore possible that at least some questions of the small selection presented above will never be answered, but answers to some of them will certainly be presented soon, because they are not only intriguing for me, but for many colleagues, too. At the same time I look forward to answers to interesting problems from other colleagues of the “sulfur family”. I use this term, because over the years, the scientists working on sulfur metabolism of higher plants have developed into a community, in which the individuals do not only know and appreciate each other scientifically, but also personally. From my point of view, this development is mostly due to Ineke Stulen and Luit De Kok, who first initiated the Sulfur Workshops, and later on started COST action 829. This is my last occasion for thanking them for all what they did to promote research on sulfur metabolism. I consider it a great privilege that I got acquainted with both of them long ago, that I could learn a lot from them and that I could feel their sympathy and friendship.
At the beginning of my work on sulfur metabolism, I was not interested in the practical application of my findings, but in basic research. I wanted to know how sulfur assimilation was regulated and where it was localized. In my grant applications, of course, I always stressed that research on plant metabolism in general and on sulfate assimilation in particular was very important, because plants are the absolute basis for nourishing men, and sulfur metabolism was not only essential for the synthesis of storage proteins, but also for coping with many stress situations encountered by plants (Rennenberg and Brunold 1994). My grant applications were accepted, I do not know, however, if the granting committees were especially impressed by my general ideas about the importance of plant metabolism.
Today, more than 800,000,000 people are suffering from hunger, and the first symptoms of climate change with its stressful consequences for plants become evident. Therefore basic knowledge about plant metabolism is more important than ever, but plant scientists have now the additional obligation to contribute to solving practical problems. In this respect, COST actions and EU projects in general represent ideal platforms, and this will even be more the case, when COST is transformed into a system, in which all participating groups are subsidized with grant money. Up to now, granting COST actions was a Swiss specialty. This made the task of the Swiss coordinator easy to bring excellent Swiss groups together for working in a COST action and the future funding will also make the job of the European coordinator a more pleasant one. At the present time, I am happy that 5 Swiss research groups, funded by the Swiss Agency for Education and Science, are working in the “sulfur” COST 829 and that two Swiss groups are participating in a “sulfur” EU project. I am especially happy about the Swiss participation in this EU project, because my successor, Doris Rentsch, joined this excellent consortium of groups.
I would like to thank my friends Luit De Kok, Dieter Grill, Malcolm Hawkesford, Heinz Rennenberg, Kazuki Saito, Ewald Schnug, Ineke Stulen and Ervin Balazs for organizing this workshop. My special thanks go to Jean-Claude Davidian, because his friendship and his kind personality gave this event a touch of warm humanity.
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