Working Group 3Metabolic engineering |
MOLECULAR COMPONENTS OF PLANT METAL ACCUMULATION: A Ca2+/Cd2+ TRANSPORTER AND NOVEL TOLERANCE GENES
Stephan Clemens, Eugene J. Kim, Dieter Neumann, Julian I. Schroeder
S. Clemens, D. Neumann, Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany.
E. J. Kim, J. I. Schroeder, Department of Biology and Center for Molecular Genetics, University of California San Diego, La Jolla, CA92093-0116, USA.
Our work is focused on identifying genes involved in the uptake of heavy metals by plant cells and their sequestration. Here we report on the identification of a transporter involved in Cd2+ uptake1 and the cloning and characterization of a novel family of metal tolerance genes from plants and yeast.
Non-essential metal ions such as cadmium are most likely taken up by plants via nutrient transporters. To identify possible pathways for Cd2+ uptake we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in S. cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more cadmium-sensitive. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 µM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements the disruption of MID1, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response . We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ across the plasma membrane and may therefore represent a first cDNA encoding a plant Ca2+ uptake pathway and may be relevant for uptake or phytoremediation of toxic metals.
To identify plant genes involved in heavy metal tolerance we used the yeast expression system. We isolated a cDNA from wheat that enables yeast cells to tolerate more than 15fold higher Cd2+ concentrations than control cells, even when expressed at low levels. This cDNA, named CdR, encodes a protein of about 55 kDa. The deduced amino acid sequence reveals no homology to proteins of known function. An Arabidopsis EST and a Schizosaccharomyces pombe ORF with homology to CdR were cloned, expressed in S. cerevisiae and were shown to also mediate Cd2+ tolerance. CdR-expressing yeast cells accumulate more Cd2+ than control cells, indicating a CdR-dependent sequestration of Cd2+ ions. However, even in mutants lacking functional vacuoles, a Cd2+ tolerance phenotype can be generated. S. pombe cells with a disruption of the CdR homolog are hypersensitive to cadmium and copper, demonstrating the physiological relevance of the CdR gene family for metal tolerance. Biochemical evidence suggests an involvement of the CdR gene products in the chelation of metal ions. CdR message is of low abundance in roots and shoots and - based on competitive PCR data - appears to be induced 5-10fold upon exposure of plants to cadmium..
1 Clemens, S., Antosiewicz, D. M., Ward, J. M., Schachtman, D. A., Schroeder, J. I. (1998) The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast. Proc Natl Acad Sci U S A. 95: 12043-8.
DETOXIFICATION OF XENOBIOTICS BY PLANTS
Julian O D Coleman
Biochemistry & Physiology Department
IACR-Rothamsted, Harpenden,
Hertfordshire AL5 2JQ
Summary: Metabolism of xenobiotics by plants: mechanisms of biotransformation and compartmentation
We are studying the biochemical processes by which foreign compounds are detoxified by conjugation to endogenous molecules (e.g. glutathione or glucose) to form water-soluble products that are then removed from the cytosol by vacuolar sequestration. Currently we are concentrating on glutathione conjugation catalysed by glutathione S-transferases and transport of the conjugates to the vacuole mediated by ATP-binding cassette (ABC) transporters. The catalytic proteins and their corresponding genes are potential sites of herbicide resistance and may have application in biological decontamination (phytoremediation).
Part of our research with the role of enhanced metabolism of herbicides as a tolerance mechanism in resistant biotypes of the weeds Alopercurus myosuroides (black-grass) and Avena spp. (wild oats). In addition we have a major link with Rhône-Poulenc Agriculture Limited to study the uptake and fate of xenobiotics in crop plants.
Key reference:Detoxification of xenobiotics by plants: chemical modification and vacuolar compartmentation, Julian O.D.Coleman, Mechteld Blake-Kalff and T.G. Emyr Davies. Trends in Plant Science (1997) 144-151.
CHARACTERISATION OF THE GENETIC AND MOLECULAR BASIS OF THE RESISTANCE OF PLANTS TO TRANSITION METALS TOXICITY
CZERNIC Pierre, FORZANI Céline, LEBRUN Michel, MARQUES Laurence
Laboratoire de Biotechnologie et Physiologie Végétale Appliquée; Université Montpellier II;
CC 002; Place Eugène Bataillon; 34095 - Montpellier - Cedex05; France.
Tel.: (33)-4-67-14-47-99; fax: (33)-4-67-14-36-37; e-mail: lebrun@arpb.univ-montp2.fr
Depending on the level of accumulation, numerous metals are either beneficial components of plant growth or poisons for plants and animals. Transition metal such as iron, copper and, to a lesser extend, nickel are among these metals. Toxic effect of these metals relies both on phytotoxicity and on accumulation in edible parts of the plants. Entry of metals in the food chain is thought to be responsible for numerous health problems. Rules on the maximum level of metal acceptable in soils have been set up to prevent entry of metals in the food chain. Restauration of soils slightly contaminated above the acceptable level can be achieved by chemical or biological tools. Phytoextraction is one of the most promising emerging biological technic for this purpose. Efficient phytoextraction relies on the capacity of plants to take up and accumulate metals. High biomass and highly resistant plants would improve the efficiency of the process. This goal needs the knowledge of the fundamental biological mechanisms that control metal acquisition and resistance by plants, as well as the molecular and cellular mechanism of resistance. Characterisation of the molecular basis of these processes would end up with plant genes that could be used either in breeding programs or for constructing transgenic plants improved in phytoextraction. Alternatively, accumulation of metals in edible parts might be manipulated.The objective of our group is to characterise the genetic and molecular basis of the resistance of plants to transition metals toxicity (iron, nickel and copper) by three main approaches:
- heterologous expression of plant cDNA in Saccharomyces cerevisiae and screening for metal-resistant phenotypes. This approach has led to the characterisation of an HMGI/Y cDNA from maize which is able to confer a specific resistance to nickel toxicity upon expression in yeast. This trait will be further assessed by overexpression of the protein in BMS maize cell suspension cultures. Other plant libraries are under screening, among which a cDNA library from Arabidopsis thaliana;
- screening of mutants of Arabidopsis thaliana on the basis of the metal-resistant phenotype. Ten thousand T-DNA insertional lines of Arabidopsis thaliana have been screened for nickel and iron resistance. Nickel resistant plants do not seem to be selectable in this screening test based on the capacity of plant to germinate and develop leaves in a metal-inhibiting medium. Iron resistant plants have been obtained and their progeny is currently being analysed. This metal-resistance screening will be soon extended to ten thousand more T-DNA insertional lines as well as to mutant lines generated by EMS. A screening based on resistance to copper toxicity of the different mutant lines has been engaged;
- use of the genetic potential of naturally resistant and hyperaccumulating plants. Genetic analysis of metal accumulation and resistance in Thlaspi caerulescens has been engaged starting from zinc and cadmium hyperaccumulating Thlaspi populations from the Montpellier’s area. Construction of cDNA libraries for expression in yeast will be soon realised from New-Caledonia endemic nickel hyperaccumulating plants: Sebertia acuminata and Psychotria douarrei.
More details will be given on the results obtained by these approaches. Perspectives and orientations will be discussed. One further objective of the presentation would be to make these approaches appealing and to extend them within our COST Action 837.
PHASE II METABOLISM OF XENOBIOTICS IN PLANTS: A MOLECULAR APPROACH
Robert EDWARDS
Crop Protection Group, University of Durham, Durham DH1 3LE, United Kingdom
Tel.: (0044) 191 374 2428; Fax: (0044) 191 374 2417; Email Robert.Edwards@durham.ac.uk
It is becoming increasingly clear that plants contain a complex array of enzymes which are able to detoxify xenobiotics including esterases and cytochrome P450-dependent oxygenases (phase 1 metabolising enzymes), glutathione transferases (GSTs), glucosyl transferases (GTs) and malonyl transferases (phase 2 metabolising enzymes) and ATP-driven vacuolar transporters (phase 3 enzymes). This enzymic complexity has been revealed by a combination of classical biochemistry and more recently through screening for putative DNA coding sequences and searches of the genome and EST databases. While molecular biology is a very powerful tool in identifying the sequences of these enzymes, it cannot yet identify their catalytic activities or functions in xenobiotic metabolism. However, we are demonstrating that the identification of putative genes encoding detoxifying enzymes can ‘fast-track’ the characterisation of complex families of enzymes which may be very difficult to study in planta due to their co-chromatography during purification, compounded by their low abundance and instability.
Using the GSTs as an example, we have identified coding sequences in cDNA libraries prepared from both crops and weeds and expressed the recombinant enzymes in bacteria for detailed assessment of their activities. We can then use a combination of antibodies raised to the recombinant proteins, HPLC fingerprinting of subunits and MALDI-TOF mass spectrometry to unambiguously identify the respective GST isoenzymes in plants. This approach has been very useful in identifying the full spectrum of GSTs in wheat and soybean, both of which contain multiple variants of GSTs due to their polyploidy.
We are now interested in collaborations with other groups leading to :
- the functional characterisation of other super-families of genes encoding detoxifying enzymes in model plants such as Arabidopsis and in crops. We are particularly interested in relating the functions of these enzymes in xenobiochemistry to their roles in endogenous metabolism. For example, we have already identified an interesting link between GSTs and oxidative stress tolerance
- an understanding of how the different components of xenobiotic metabolism in plants function together, and in particular how they are differentialy regulated by biotic and abiotic stress
- new methods for identifying enzymes for which we have no leads as to their likely coding sequences.
GLUTATHIONE BIOSYNTHESIS IN HIGHER PLANTS: MOLECULAR AND PHYSIOLOGICAL APPROACHES.
Laurence Gondet, Pascaline Ullmann and Thomas J. Bach.
Laboratoire d’Ecotoxicologie Végétale, Institut de Biologie Moléculaire des Plantes (CNRS-UPR 406) - Institut de Botanique, 28 rue Goethe, 67083 STRASBOURG Cedex, FRANCE.
P.Ullmann@ibmp-ulp.u-strasbg.fr
The structure of glutathione (GSH, -glu-cys-gly) is well suited to an aerobic environment. Among variuos the functions ascribed to this tripeptide, its early role seems to be a safe and stable storage form of cysteine. To better understand how GSH biosynthesis has evolved and is regulated, cloning of the corresponding genes is required. GSH is synthesized in two sequential ATP-dependent reactions, catalysed by g-glutamylcysteine synthetase (GSHI) and glutathione synthetase (GSHII).
We focused first on isolation and characterization of GSH1 and GSH2 Arabidopsis thaliana cDNAs by functional complementation of GSH-deficient yeast mutant strains. We constructed also gene-disrupted yeast strains (gsh1 ::HIS3 and gsh2 ::HIS3), which will allow the overexpression of the plant cDNAs for molecular and biochemical analyses. Moreover, the null gsh1 strain shows an interesting phenotype, which could serve to isolate GSH1 from other organisms. This phenotype should also provide a way to examine how the intermediate -glu-cys could replace GSH for some essential functions.
At the same time, we studied the interactions between the dichloroacetamide-type herbicide safeners (also called herbicide antidotes) and toxic heavy metals in maize (Zea mays), in relation to their effects on GSH metabolism. Our morphological, biochemical and cytochemical observations revealed an important protective effect of these safeners against cadmium toxicity in maize roots, caused by an overproduction of GSH, leading to a greater amount of phytochelatins (PCs), which in turn allows the sequestration of heavy metals.
Furthermore, we started a program of stepwise selection of A. thaliana cells, resistant to buthionine sulfoximine (BSO), which is a specific inhibitor of GSHI. Our results indicate that BSO tolerance in the selected cell lines is correlated with an increased level of GSH. Yeast and human GSH1 genes are regulated by transcription factors like AP-1. Whether such mechanisms occur in BSO tolerant Arabidopsis cells needs further investigation.
UNDERSTANDING METAL HYPERACCUMULATION IN PLANTS
Ute Krämer
Plant Physiology and Biochemistry Department, Fakultät für Biologie IV- W5, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany,
ute.kraemer@biologie.uni-bielefeld.de
Some plant species are known to accumulate large concentrations of metallic cations in their above-ground biomass. Metal concentrations in the shoots of these so-called metal hyperaccumulators are usually at least one, often several orders of magnitude higher than those in surrounding plants on the same, metal-rich soils. Thus metal hyperaccumulators have been defined as containing at least 1000 µg g-1 Ni, Co, Cu or Pb or more than 10,000 µg g-1 Zn or Mn in above-ground tissues of field-collected specimen on a dry biomass basis. The ability to accumulate metal cations to very high levels specifically in the shoot without suffering from metal toxicity is highly desirable for plants used in the cleanup of metal-contaminated soils. However, the genetic and biochemical basis of hyperaccumulation and associated metal tolerance are poorly understood to date.
About four hundred metal hyperaccumulator species are currently known, 75 % of which are hyperaccumulators of nickel. In the Ni hyperaccumulator Alyssum lesbiacum exposure to Ni resulted in a large and proportional increase in the concentrations of free histidine in xylem sap and both root and shoot tissues. Histidine is capable of selectively chelating Ni and may thus reduce the amounts of cytosolic free Ni which could inactivate crucial metabolic sites. It was shown by EXAFS (Extended X-ray Absorption Fine Structure) analysis that histidine, and not sulphur, is indeed an important Ni ligand in planta. When the related non-tolerant non-accumulator A. montanum was provided with exogenous histidine, Ni tolerance and Ni translocation rate from root to shoot were increased significantly. This suggests that in the hyperaccumulator A. lesbiacum the production of free histidine is involved in both Ni tolerance and root-to-shoot translocation of Ni. Recently, plant cDNAs have been identified for all steps involved in histidine biosynthesis.
Metal hyperaccumulators differ in metal specificity and extent of hyperaccumulation. Thus it is likely that several mechanisms have evolved leading to hyperaccumulation of the respective metals. Our aim is a molecular analysis of Ni and Zn hyperaccumulation and ultimately, to model these processes in a transgenic approach.
THE POTENTIAL APPLICATION OF PLANT CYTOCHROME P450S IN ORGANIC POLLUTANT DETOXIFICATION
F.A. Krens
Department of Cell Biology, CPRO-DLO
P.O. Box 16, 6700 AA Wageningen, The Netherlands
Cytochrome P450s represent a class of enzymes that are present in most organisms and are involved in different types of oxygenation reactions using a variety of substrates. Amongst others, pesticides and other organic pollutants can serve as substrates. Substrate specificity can be broad in some enzymes as well as very limited in others. At CPRO-DLO there is an interest to investigate potential applications of cytochrome P450s in phytoremediation by detoxifying environmental pollutants.
At present 25 to 30 plant cytochrome P450 encoding genes have been isolated and identified from several plant species in ongoing gene isolation projects using random sequencing and BLAST searches. It is envisaged that this number will increase further in the near future. Plans are to prepare fusion proteins between the plant cytochrome P450 and an electron donor, NADPH cytochrome P450 reductase, express them in heterologous systems and to determine their detoxification capacity in a high through-put system testing a range of organic pollutants. Genes of potentially interesting enzymes can be used in genetic engineering of target plants for phytoremediation or other organisms for bioremediation. Also application in cell-free systems after immobilization will be considered.
COST 837 WORKING GROUP 3 : CONTRIBUTION OF THE LABORATORY FOR PLANT BIOTECHNOLOGY ICAT
M. Salomé PAIS
Laboratory of Plant Biotechnology ICAT, Ed. ICAT, 1749-016, Lisboa, Portugal
For about 15 years now the Laboratory of Plant Biotechnology is involved in different approaches using woody plants as plant material. It is currently mostly engaged in gene isolation and genetic manipulation of woody species such as poplar, European chestnut, pears and tropical fruit species such as avocado, mango and papaya. However, other important crops and plants used in floriculture have been or are still being studied, aiming at their genetic manipulation in ordere to get better characteristics.
The main approaches used at the laboratory are based on current techniques of molecular biology:
- DNA and RNA isolation, construction of cDNA libraries, DDRT-PCR techniques, RAPDs, APLPs and microsatellites, Southern, Northern and Western Blotting, in situ hybridization;
of genetic manipulation:
- direct gene transfer to protoplasts, to organs or calli by particle bombardment and by vacuum infiltration and Agrobacterium-mediated transformation; sense and antisense technologies.
The Laboratory of Plant Biotechnology is willing to contribute to WG3 whenever this expertise can be used.
We will report in detail on the most interesting results taking into account the aim of WG3.
In terms of gene isolation and cloning, several sequences have been submitted to the gene data bank; others are still kept confidential due to their potentially commercial interest.
Some examples of genetic manipulation will also be reported.
ISOLATION AND CHARACTERIZATION OF GENES FOR RESISTANCE TO CHEMICAL STRESSES
M. Sari-Gorla, C. Frova, E.M. Pè
Department of Genetics and Microbiology, University of Milano
Via Celoria 26, 20133 Milano, Italy
The final goal of our researches in the field of plant tolerance against chemical stresses is the development of genotypes able to cope with stresses due to the presence of xenobiotics. The compounds we studied so far are herbicides.
In order to achieve this purpose, we have followed two strategies. The first approach concerns the development of new strategies of selection: gametophytic selection (selection at pollen level), and genetic dissection of plant tolerance by linkage analysis with molecular markers, in order to identify and localize on chromosomes the genetic factors conferring tolerance. This information can be used for marker assisted selection (MAS), a procedure much more powerful than traditional breeding methods. Results of this work were the production of improved maize genotypes and the detection of QTLs in the same species, which explain a large proportion of plant variability for tolerance to the herbicide Alachlor.
A second approach is based on the detection of tolerance mechanisms, in particular the mechanisms for detoxifixation of xenobiotics, and the identification of the genetic system controlling them. Since some years, our interest is pointed to the system glutathione/glutathione S-transferase (GSH-GST), which plays and important role in the detoxification of several toxic molecule: GSTs are involved in the response to different stresses and have precise pattern of expression. Different aspects of the genetic and molecular characterization of the Gst gene family in maize were carried out. Three of the genes have been localized on maize chromosomes. The functional characterization of the different isoforms allowed their possible role in plant defence against herbicide to be defined. Furthermore, expression analysis, at transcript and gene product level, indicated that the isoforms are developmentally regulated and that some of them are induced by specific herbicides.
At present we are studying the plant response to different stress factors: alcohols, heavy metals (cadmium), tiocarbamates, in maize, and we intend to characterize the Gst gene family in rice. The choice of this species is due to its agronomic importance and, at the same time, to its amenability for biotechnology and its simple genome, so that it can be considered a model species for cereals.
The results obtained in maize and rice will be used to transfer the information to species not genetically well known, but having useful characteristics under the aspect of xenobiotic resistance. One of these is Brassica juncea (Indian mustard). This species its able to cope very well with the presence of heavy metals in the soil, which the plant is able to accumulate, removing them from the environment. Thus, the characterization at genetic and molecular level of the biological system of resistance in this species should provide important tools in the field of phytoremediation.
SULFUR ASSIMILATION AND GSH BIOSYNTHESIS IN THE HEAVY METAL ACCUMULATOR BRASSICA JUNCEA L.
Holger J. Schäfer, Senta Heiss, Angela Haag-Kerwer and Thomas Rausch
Botanisches Institut, INF 360, D-69120 HEIDELBERG, Germany
trausch@botanik1.bot.uni-heidelberg.deBrassica juncea is a high biomass crop with potential for phytoremediation of heavy metal-polluted soils. When exposed to Cd via the root system, a strong induction of phytochelatin (PC) synthesis is observed in roots and shoots alike. Since PCs are synthesized from glutathione (GSH), we have investigated the molecular physiology of sulfur assimilation and GSH biosynthesis in Cd-exposed plants. cDNAs have been cloned for a low affinity sulfate transporter (LAST), and for isoforms of ATP sulfurylase (ATPS), APS reductase (APSR) and O-acetylserine (thiol)lyase (OAS-TL), as well as for g-glutamylcysteine synthetase (ECS) and glutathione synthetase (GSHS) [1,2]. These cDNAs have been expressed in E. coli and P. pastoris, and antisera have been raised against the recombinant proteins. With these tools, we are studying the expression of these genes at the transcript and the protein level. In particular, we explore their regulation in response to nutrient status (S/N) and various stress treatments, including heavy metal exposure. In parallel, we determine cellular concentrations of cysteine, GSH and PCs by HPLC analysis. As a major result of our studies we could show a coordinate induction of ATPS, APSR [2] and ECS [1] in response to Cd-exposure. Thus, massive PC formation appears to depend on up-regulation of sulfur assimilation and GSH biosynthesis. Interestingly, in roots Cd caused a repression of LAST [2]. The physiological role of the cloned LAST isoform is not yet known, but it has been hypothesized that this transporter could be involved in root-shoot transfer of sulfate [2]. Immunocytochemical and in situ hybridization techniques will now be used to map the spatial distribution of gene expression in roots and leaves. Furthermore, subcellular fractionation studies are under way to localize different isoforms. Thus, ECS polypeptides have been detected in the cytosol, the chloroplast/plastid fractions and in peroxisomes [3], suggesting that different organelles may be autonomous with respect to GSH biosynthesis. By rigorously defining the key regulatory steps for GSH biosynthesis during long-term exposure to Cd (or other stress factors), we aim to identify targets for genetic manipulation to improve the heavy metal-sequestering capacity and/or stress tolerance of Brassica juncea.
- Schäfer, H.J., Haag-Kerwer, A. and Rausch, T. (1998): cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial g-glutamylcysteine synthetase isoform. Plant Mol.Biol. 37: 87-97
- Heiss, S., Schäfer H.J., Haag-Kerwer, A. and Rausch, T. (1999): Cloning sulfur assimilation genes of Brassica juncea L.: Cadmium differentially affects the expression of a putative low affinity transporter and isoforms of ATP sulfurylase and APS reductase. Plant Mol.Biol. in press
- Schäfer, H.J. and Rausch, T. (1999): Higher plant g-glutamylcysteine synthetases: functional complementation of a yeast mutant and subcellular localization of isoforms. submitted
AN ARABIDOPSIS ABC TRANSPORTER EXHIBITING GLUTATHIONE-CONJUGATE AND CHLOROPHYLL CATABOLITE TRANSPORT ACTIVITY
Roberto Tommasini1, Esther Vogt1, Stefan Hörtensteiner2 Markus Klein3 and Enrico Martinoia3
1Institute of Plant Sciences, Swiss Federal Institute of Technology
2Department of Plant Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland
3Université de Neuchâtel, Laboratoire de Physiologie Végétale, Rue Emile Argand 13, 2007 Neuchâtel, Switzerland
Metabolism of herbicides to their glucosides or GSH conjugates is usually considered a detoxification process, but these products may exert other biological activities. Efficient detoxification requires therefore excretion of the glutathione conjugate into the vacuole, an organell with limited metabolic activity.
The tonoplast contains two proton pumps, an H+-ATPase and H+-PPase that establish and maintain an electrochemical gradient that can be used for secondary activated transport processes. Surprisingly, glutathione conjugate uptake was not energized by the vacuolar proton pumps but rather by a specific glutathione-conjugate pump. Accumulation of glutathione conjugates within the vacuole occurred even when the vacuolar ATPase was inhibited and the DpH between the cytosol and the vacuole was abolished. Treatment of barley plants with so-called safeners or Arabidopsis thaliana with chlorodinitrobenzene, which is conjugated to glutathione within the cell , increases the activity of the glutathione conjugate pump.
Testing available yeast strains devoid of individual ABC transporters, for their capacity to transport glutathione conjugates revealed that a cadmium-sensitive yeast strain with a deletion in the ycf1 gene had a strongly reduced glutathione conjugate transport activity (10 to 20% of the control value), indicating that the gene product of ycf1 is a glutathione conjugate transporter. YCF 1 is highly homologous to another member of the ABC family, namely the human MRP (multi drug resistance associated protein). Heterologous expression of MRP in the ycf1 mutant restord the glutathione transport activity. Furthermore, ycf1 mutants expressing MRP were again tolerant to cadmium (Tommasini et al, 1996).
A database search for plant proteins homologous to MRP1 and YCF1 revealed that at least four proteins of Arabidopsis thaliana are highly similar to MRP1 and YCF1 and, therefore, must be considered putative glutathione-conjugate transporters. In an attempt to circumstantially assign a function to the different MRP-like proteins, expression studies using plants treated with xenobiotics known to be detoxified through conjugation to either glutathione or glucose, were performed, but did not reveal a correlation between increased transcript levels and the respective detoxifying pathway. However, the level of the transcript of one of the gene coding for a MRP-like protein was found to be stongly increased in response to treatment with primisulfuron, a sulfonylurea herbicide inhibiting branched chain amino acid biosynthesis, and with IRL 1803, a triazine inhibiting the histidine biosynthetic pathway (Tommasini et al. 1997). The respective gene was cloned and expressed in the yeast ycf1 mutant. Cadmium tolerance and glutathione conjugate transport activity were partially restored. Surprisingly, the plant gene product was also able to transport chlorophyll catabolites (Tommasini et al. 1998). Such a double activity was also observed for another Arabidopsis MRP-like transporter (Lu et al. 1998). Presently we are analyzing in more details this Arabidopsis ABC transporter and we produced transgenic plants containing the promotor-GUS construct.
Tommasini, R., Evers, R., Vogt, E., Mornet, C., Zaman, G.H., Schinkel, A.H., Borst P. and Martinoia, E. (1996) Proc.Natl. Acad. Sci. USA 93, 6743 - 6748
Tommasini, R., Vogt, E., Schmid, J., Fromenteau, M., Amrhein N. and Martinoia, E. (1997) FEBS Lett. 411, 206 - 210
Tommasini, R., Vogt, E., Fromenteau, M., Hörtensteiner, S., Matile, P., Amrhein, N. and Martinoia, E. (1998) Plant J. 13, 773 - 780
Lu, Y.P., Li, Z.S., Drozdowicz, Y., Hörtensteiner, S., Martinoia E. and Rea P.A. (1998) Plant Cell 10, 267 - 282
PLANT-BACTERIUM COMBINATIONS FOR EFFECTIVE REMOVAL OF ORGANIC POLLUTANTS
Peter J. Weisbeek, Alex Ooyevaar and Han Gerrits
Department of Molecular Genetics, University of Utrecht, Utrecht, The NetherlandsThe aim of our research is to explore the potential of the plant rhizosphere for the degradation of soil pollutants and to develop cleanup methods based on the degradation capacity of the rhizosphere. Two parallel research lines are followed. The first one addresses the enhanced growth of degrading Pseudomonas strains on the plant root. The second line focuses on the activation in the plant of specific degradative and general stress responses by xenobiotics and root-colonising bacteria. The rhizosphere, the volume of soil that surrounds the root and is influenced biologically and physically by the living root, is an important interface for microbial and microbe-plant interactions, with continuous exchange of informational signals and adaptation to local conditions. Efficient root colonization by micro-organisms is essential for symbiotic and pathogenic interactions and for the successful use of microbes in processes such as biocontrol, biofertilization and phytoremediation. The density of specific soil bacteria is increased enormously in the rhizosphere; bacterial cell numbers can be 1,000- to 10,000-fold higher than in plantless soil. The difference is mainly due to the constant supply of nutrients in the form of plant exudates, which constitutes up to 35% of the total photosynthate of the plant.
An important prerequisite for microbial growth in soil and rhizosphere is the acquisition of iron. A complex and intriguing siderophore-dependent iron uptake and signal transduction system in Pseudomonas putida WCS358 was analysed in detail and found to provide the strain with a strong competitive advantage. This property was optimised and subsequently linked to the ability to degrade aromatic compounds such as toluene, xylene, naphtalene and phenantrene by mobilisation of degradative plasmids into these colonising strains. The degradative capacity of the resulting strains is being monitored in vitro and in situ.
The close interaction between plants and microbes in the rhizosphere suggests the possibility that plants and microbes have evolved mechanisms to influence each other's behaviour and metabolism. A number of signals and mechanisms have recently been identified that support this idea; e.g. one of the responses associated with iron limitation in the rhizosphere is the activation of specific plant defence pathways. Our second line of research builds on this potential; it focuses on the activation of plant genes that are necessary for xenobiotic degradation and for growth of the plant under less favourable conditions. We found that selected rhizosphere bacteria can activate specific plant defence pathways. Analysis of bacterial signals and induced plant genes opens the way to control plant gene expression through rhizosphere bacteria, with the genes for degradation and survival as attractive targets.
A combination of successful bacterial growth and properly activated plant metabolism is expected to have good potential for in situ degradation of xenobiotics.
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