Working Group 3
Dipartimento di Genetica e di Biologia dei Microrganismi
University of Milano
Via Celoria 26, Milano, Italy
Local organizer: Prof. Mirella Sari-Gorla
Friday, December 15
16:00 David Dixon (GB)
Harnessing natural and forced evolution for the detoxification of organic pollutants
16:30 Michel Lebrun (FR)
Effect of nichel on chromatin condensation and resistance conferred by the hmgi/Y protein of maize
17:00 Enrico Martinoia (CH)
Vacuolar transporter involved in heavy metal resistance
17:30 G. Mavrogianopoulos, S. Kyritsis
Efficiency of Giant reed (Arundo donax L.), as a biofiltering device for sewage effluents
18:00 Thomas Rausch (DE)
Regulation of GSH synthesis in B. juncea under stress
18:30 General discussion
Saturday, December 16
9:30 Nicole Soranzo (IT)
A genomic approach to the analysis of the GST multigene family
10:00 Arja Tervahauta (FI)
Plant metallothioneins in metal detoxification
10:30 Coffee break
11:00 Gian Attilio Sacchi (IT)
Cd-induced sulphate uptake in maize root
11:30 Poster discussion:
Valerie Bert, Pierre Saumitou-Laprade, Nathalie Verbruggen (BE)
Identification of genes involved in cadmium tolerance and hyperaccumulation in Arabidopsis halleri
Maura Cardarelli (IT)
Overexpression of phytochelatin synthase in hairy roots of Nicotiana tabacum
12:00 General discussion
David P. Dixon, David J. Cole* and Robert Edwards
Department of Bioligical Sciences, University of Durham, Durham, UK.
*Aventis CropScience UK Ltd., Ongar, Essex, UK.
Phytoremediation of many organic pollutants requires enzymes capable of degrading these compounds.Such enzymes can be found by searching for activities among plant enzymes capable of related biotransformations, and then improved by altering such enzymes to shift their activities towards the required substrates.Glutathione transferases (GSTs) in plants are ideal phytoremediation enzymes as they are easy to work with and their diversity and broad activities mean that they are capable of degrading a huge range of organic pollutants.
We have therefore examined plant GSTs potentially able to be used for phytoremediation, investigating the natural diversity of these enzymes to identify isoforms with useful activities. The activities of these chosen enzymes towards organic pollutants have then been further enhanced using directed evolution, where random mutations have been introduced and enzymes with enhanced activity have been selected from the resulting pool of mutants. Using this approach we have identified maize GSTs with high activity towards diphenylether herbicides. Subsequent directed evolution and site-directed mutagenesis further increased the enzymes activity 30-fold towards the chosen substrate, confirming that this technique can engineer enzymes able to rapidly degrade organic pollutants.
Céline Forzani, Clarisse Loulergue, Stéphane Lobréaux, Jean-François Briat and Michel Lebrun
Biochimie et Physiologie Moléculaire des Plantes. Centre National de la Recherche
Scientifique (Unité Mixte de Recherche 5004), Université Montpellier 2, Institut National
de la Recherche Agronomique, Ecole Nationale Supérieure dAgronomie, 2 place Viala,
F-34060 Montpellier cedex 1, France
Expression of a maize cDNA encoding an HMG I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the void vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains 4 AT-hook motifs known to be involved in binding to the minor groove of AT rich DNA regions. HMG I/Y proteins may act as achitechtural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-GFP fusion protein was observed in the yeast nuclei.
Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel, and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild type level of nickel sensitivity to the Drpd3 yeast mutant. Therefore, resistance to nickel of yeast cell expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.
Expression analysis of some members of the AtMRP subfamily in response to cadmium
Lucien Bovet and Enrico Martinoia
Laboratoire de Physiologie Végétale, Université de Neuchâtel, Rue Emile Argand 13, 2007 Neuchâtel
The abstract is not available. Please contact the authors for further information.
Efficiency of giant reed (arundo donax l.), as a biofiltering device for sewage effluents
G. Mavrogianopoulos and S. Kyritsis
Agricultural University of Athens, Athens Greece
One experimental field has been established in Central Greece close to a pig-raising farm. Two Giant reed populations were cultivated in a closed gravel hydroponic system, where nutrient solution was pigs sewage effluent. The amounts of all nutrients in the pigs sewage effluent were rather high, compared with plant uptake, except for phosphorous that was rather low.
The results showed that Giant reed is a high yielding non-food crop and simultaneously an efficient plant for biofiltering sewage effluents. Water evapotranspiration during the first, second and third year was 500, 400 and 480 lm-2yr-1 respectively. According to stem analysis, for the first and second year, there was an average infiltration rate of 31g m-2 yr-1 total N, 7.5 g m-2 yr-1 total P, 18.8 g m-2 yr-1 K, 2.1 g m-2 yr-1 Ca, 2.1 g m-2 yr-1 Mg, 0.27 g m-2 yr-1 Fe, 0.02 g m-2 yr-1 Mn, 0.14 g m-2 yr-1 Zn and 0.08 g m-2 yr-1 Cu. During the third year, when wastewater with the current nutrient and metal content improved with some extra P, the average infiltration rate for most elements increased by 46% and for P by 169%. This is a result of a higher biomass production and higher P concentration in the stem tissues. Concentrations of nutrients N, K, Mg, Ca, Mn, Fe, in plant tissues were at the same levels as the ordinary grown plants in the soil. Only P, during the first two years, was accumulated in the plant tissues in a lower concentration compared to the ordinary grown plants in the soil due to low amounts of P in the wastewater. Wastewater trace elements Cu and Zn in general accumulated in the plant tissues in a higher concentration compared to the ordinary grown plants in the soil.
In general, Giant reed cultivated in a close gravel hydroponic system, where nutrient solution was wastewater, is contributing to the possibilities of designing sustainable systems for wastewater use in a productive way.
Key words: Giant Reed, Arundo donax, wastewater, biofiltering, biomass, reuse, hydroponic, pigs sewage effluent
Regulation of glutathione synthesis under stress
Senta Heiss, Andreas Wachter, Holger Schäfer, Jochen Bogs, Aletta Wilhelm and Thomas Rausch
Botanical Institute, University of Heidelberg, Germany
Brassica juncea is a high biomass crop which has potential for bioremediation of heavy metal-polluted soils. As the formation of phytochelatins (PC) is an essential component of cellular detoxification, we are investigating the regulation of S-assimilation, gluathione (GSH) synthesis, and PC formation under different stress regimes. In particular, we are studying i) the coordinate up-regulation of gECS and S-assimilation genes after Cd exposure, ii) the compartmentation of GSH synthesis under different stress regimes, and iii) the expression of PC Synthase in response to Cd exposure.
For the genes of interest, we have cloned cDNAs and expressed the recombinant proteins in E. coli for antiserum production. For gECS targeting studies, we have cloned several transit peptide-RFP (red fluorescent protein) fusions and used them for transient transformation via particle bombardment. Our results show that Cd induces a coordinate increase of transcripts for several genes involved in S-assimilation and GSH synthesis, however, the induction of mRNA is not always leading to an increase of the corresponding protein. Furthermore, different stress regimes (Cd exposure, wounding or JA treatment) affect the compartmentation of GSH synthesis; while the cytosolic gECS isoform is slightly up-regulated the chloroplast isoform is strongly down-regulated. Finally, we could show that the PC synthase protein is up-regulated after prolonged Cd exposure.
Our results demonstrate the complexity of the metabolic adaptation during PC synthesis in response to different stress regimes. Further elucidation of the functional and regulatory links between increased cytosolic GSH demand and adaptation of gene expression and/or compartmentation will require an integrated approach, involving multiparallel gene expression analysis (genomics/proteomics), metabolite analysis, and further study of stress-induced changes in subcellular compartmentation.
[This research is supported by the DFG: Forschergruppe 383 & Graduiertenkolleg 388].
A genomic approach to the analysis of the GST multigene family
Nicole Soranzo, Carla Frova, M.Enrico Pè, Luca Mizzi, Ester Boldrighi, Diana Santelia, Mirella Sari-Gorla
Department of Genetics and Microbiology
University of Milano
Understanding of the biological mechanisms underlying the response to chemical pollutants in plants may provide us with cheap and environment-friendly tools to address the effect of increasing anthropogenic pressure in many areas of the world. Among the many enzyme systems involved in the detoxification of toxic compounds, the family of glutathione S-transferases (GSTs) represents a particularly appealing study system due to its ubiquitous expression and broad substrate specificity. Glutathione-S-transferases catalyse the addition of the tripeptide glutathione to toxic electrophilic compounds to form inactive conjugates; other enzymatic activities include the glutathione-dependent peroxidase, esterase and isomerase. The GST activity is induced in response to various sources of biotic and abiotic stress, including pathogen attack, chemical stress due to xenobiotic compounds and oxidative stress caused by heavy metals.
Our main research interest is to investigate the role played by different glutathione-S-transferase enzymes in the response to various sources of chemical stress. In this paper, we perform a comprehensive survey of the GST multigene family in the two model species Oryza sativa and Arabidopsis thaliana. The isolation of GST genes was pursued for both species using a bioinformatic approach. The isolation of Arabidopsis genes was obtained exploiting both the genome sequence information and the EST collections. Redundant clones were eliminated, yielding to about 50 different putative GST-encoding sequences. The isolation of rice genes was obtained by screening the EST sequence databases using consensus aminoacidic sequences representing conserved GST domains of the two main plant phylogenetic classes Phi and Tau. Using this approach, 187 different EST clones were identified as putative GSTs, encoding for at least 35 different gene products. Plasmids for 36 of them were obtained and sequencing analysis is currently under way. So far, eighteen genes have been isolated, all representing full-length clones encoding for proteins of approximately 220 aa; we estimate that at least 35 putative rice GST genes will be isolated using this method. Phylogenetic analysis on the aminoacidic and deduced aminoacidic rice and Arabidopsis sequences showed that the majority of the genes belong to the three main plant phylogenetic classes Phi, Tau and Zeta. Pairwise intra-class aminoacidic identities range between 50 and 75%, whereas figures lower that 35% were obtained for inter-class comparisons.
Expression analysis on nine rice genes showed that all but one of the genes analysed are constitutively expressed in root, shoot, seedling and inflorescence tissues. Treatment of hydroponically-grown plants with various agents including cadmium and three different classes of herbicides was used to characterise the specificity of GST expression in conditions of chemical stress.
Plant metallothioneins in metal detoxification
Arja Tervahauta1, Viivi Hassinen1, Nathalie van Hoof2, Henk Schat2, Jos Verkleij2, and Sirpa Karenlampi1
1Department of Biochemistry, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland 2Department of Ecology and Ecotoxicology of Plants, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Metallothioneins (MTs) are low-molecular-weight, cysteine-rich cytoplasmic metal binding proteins that could protect cells against the toxic effects of metals by chelating them. Genes encoding MTs occur in animals, higher plants, eukaryotic microorganisms and in some prokaryotes. There are different MT protein families, subfamilies, subgroups and isoforms. Metallothionein genes of mammalians, yeast and plants have been transferred in plants resulting increased tolerance to Cd with a minor effect to metal accumulation properties in plant. Metal-controllable metallothionein promoters from mammalians and yeast have been used in plant transformation.
The functions of MTs in plants are still unclear. However, Murphy and Taiz (1995) found that MT2 gene expression was the primary determinant of ecotypic differences in the copper tolerance of Arabidopsis seedlings. Moreover, (over)expression of MT genes in Escherichia coli or in MT deficient yeast indicated for a role of MTs in heavy metal tolerance in plants (Zhou and Goldsbrough, 1994). For these reasons, it is conceivable that MTs might play a role in copper tolerance in Silene vulgaris.
Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance (Schat et al. 1996; Schat and Vooijs 1997). To evaluate the role of metallothioneins (MTs) in copper tolerance in Silene vulgaris, we screened a cDNA library derived from a highly copper tolerant Imsbach population, using Arabidopsis-based MT probes and identified a MT2b-like gene (Tervahauta et al. 1998, Genebank Accession No AF 101825). Northern blot analysis showed that plants from the copper tolerant Silene vulgaris Imsbach population had much higher transcript levels of this gene, SvMT2b, than those from the copper sensitive Amsterdam population. This was shown for both roots and shoots of copper-exposed and nonexposed plants. When expressed in yeast, SvMT2b restored cadmium and copper tolerance in different metal sensitive strains. Segregating F3 families of crosses between copper sensitive and copper tolerant plants showed a 1 to 3 segregation for SvMT2b expression. Allele specific PCR showed that low expression F3 plants were homozygous for the allele inherited from the copper sensitive parent, whereas high expression plants were either heterozygous or homozygous for the allele derived from the tolerant parent. SvMT2b
expression did not co-segregate with copper tolerance in the F3 families. However, a significant co-segregation did occur in F4 families derived from crosses between moderately tolerant F3 plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b produced copper tolerance, though only within the genetic background of a copper tolerant plant. This suggests that the regulation of the expression of SvMT2b has a role in copper tolerance. We have isolated a 283 bp fragment upstream from the transcription initiation site of SvMT2b. Comparison of this sequence in Cu tolerant and sensitive plants showed differences only in two nucleotides. Studies are planned to further explore the regulation of SvMT2b. A metal-responsive promoter system could find use in plants designed for phytoremediation.
Murphy A, Taiz L (1995) Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Plant Physiol 109: 945-954
Schat H, Vooijs R, Kuiper E (1996) Identical major gene loci for heavy metal tolerances that have evolved in different local populations and subspecies of Silene vulgaris Evolution 50: 1888-1895
Schat H, Vooijs R (1997) Multiple tolerance and co-tolerance to heavy metals in Silene vulgaris: a co-segregation analysis. New Phytol. 136: 489-496
Zhou J, Goldsbrough PB (1994) Functional homologues of fungal metallothionein genes from Arabidopsis. Plant Cell 6: 875-884
Cd-induced sulphate uptake in maize roots
Livia Pirovano, Fabio Nocito, Maurizio Cocucci and Gian Attilio Sacchi
Department of Plant Production, University of Milan
Use of plants in phytoextraction of heavy-metals (HMs) is based not only upon their ability to take up, accumulate and translocate these xenobiotics, but also upon a complex pool of mechanisms able to alleviate their toxic effects. The knowledge of these topics is crucial in the basic understanding of tolerance, for improving plants of biotechnological interest. In higher plants cadmium exposure induces the synthesis of phytochelatins (PCs) from glutathion (GSH) by phytochelatin synthase. PCs forms complexes with cadmium reducing its activity in the cytosol. PCs biosynthesis is strictly related to the sulphur metabolism. Many enzymes involved in the sulphate reductive assimilation pathway and GSH biosynthesis are induced following Cd exposure, indicating a cellular response to transient GSH depletion during phytochelatin production. The general alteration of the sulphur metabolic pathways is a probable consequence of an increase in GSH demand, driven by PCs biosynthesis. In other words, cadmium exposure would induce an additional sink able to increase the need of thiol-compounds by plants. In this context, it is of particular importance to study the regulation of transport systems involved in sulphate uptake from the soil solutions by roots.
Maize root seedlings exposed to 10 µM CdCl2 during the growth showed a two-fold increase in SO42- influx measured in short-time experiments at 200 µM external SO42-. This effect was detectable after 24 h of Cd exposition and was maintained up to 72 h. In the same condition the influx of other nutrients was negatively (K+ and Cl- ) or not (PO43-) affected. The higher SO42- influx was not a consequence of an enhancement in the transmembrane H+ electrochemical gradient that energises it. In fact, the transmembrane electric potential difference resulted more negative in the cortical cells of the treated root and moreover the level of PM H+-ATPase (determined by both Northern and Western analysis) and its actvity (measured on purified plasma membrane vesicles) resulted negatively affected by the treatment. Northern blot experiments showed a close relationship between SO42- uptakes and the expression level of a root mRNA codifying for a putative high-affinity sulphate transporter. Total sulphur content of roots and shoots of Cd-exposed seedlings was higher than those of the control, according to the SO42- transport activities described above. Differently the SO42- content of the roots dramatically decreased in the early phases of Cd exposure. This effect could be due to the well described Cd-induced activation of the sulphate assimilatory pathway and could be involved in the transcriptional control of the gene encoding for high-affinity sulphate transporter.
In maize roots Cd-exposure induces adaptive responses including a positive pretranslational regulation of the gene codifying a high affinity sulphate transporter and a consequent enhancement of sulphate influxes. We suggest that this response is triggered by the enhanced need of thiol-compounds necessary for metal detoxification, rather than a non specific effect due to Cd accumulation. The efficiency of the response and above all its synchronisation with the increasing level of Cd in the cells might play a role in the tolerance of the plant to the HM.
Identification of genes involved in cadmium tolerance and hyperaccumulation in arabidopsis halleri
Bert1, Pierre Saumitou-Laprade2
and Nathalie Verbruggen1
1Laboratoire de Physiologie et de Génétique Moléculaire des Plantes, Université Libre de Bruxelles (ULB), CP 242, Boulevard du Triomphe, 1050 Bruxelles, Belgium.
2Laboratoire de Génétique et Evolution des Populations Végétales, UPRESA 8016, Université de Lille 1, 59655 Villeneuve dAscq, France
Heavy metals are highly toxic to plants. Only tolerant plants, which are called metallophytes, can grow and reproduce on metal-contaminated sites. Very few of them hyperaccumulate metals, i.e. translocate significant quantities of metal to their shoots. Very little is known about the molecular genetics of tolerance or hyperaccumulation. The aim of the project is to identify genes involved in tolerance and/or hyperaccumulation of cadmium, which is one of the most aggressive metals for plants.
To identify those genes, segregating populations from crosses between a tolerant/ hyperaccumulating species and a non-tolerant/non-hyperaccumulating species will be used. Arabidopsis halleri hyperaccumulates Cd. A. halleri is closely related to and interfertile with Arabidopsis petraea subsp. Lyrata, which is both non-tolerant to Cd and a non-accumulator. Both plants are easy to propagate by cutting. Successful crosses have been made between A. lyrata and A. halleri (Saumitou-Laprade, unpublished). Preliminary results showed that the EC100 of A. halleri is 75 mM Cd and the one of A. lyrata is 10 mM Cd. A F1 progeny was back-crossed to A. lyrata subsp petraea plants. Cuttings from 100 plants are being analysed for their tolerance to Cd (by EC100 determination) and hyperaccumulation (by ICP analysis). According to the EC100 results of the grandparents, a clear segregation is expected in the progeny of the back-cross.
We aim at identifying genes associated with Cd tolerance and/or hyperaccumulation in A. halleri. Transcript patterns of the above-described back-cross, will be studied by cDNA-AFLP (Amplified Fragment Length Polymorphism), a technique which is highly sensitive and robust. Using presence and abundance criteria, the transcripts (actually, the transcript derived DNA fragments visualised in the cDNA-AFLP analysis), the abundance or presence of which are correlated with Cd tolerance and/or hyperaccumulation will be further cloned and characterised. This study may significantly contribute to the identification of Cd tolerance and hyperaccumulation mechanisms.
Overexpression of phytochelatin synthase gene in nicotiana tabacum 'hairy roots'
Mirella Pomponi1, A. De Paolis2, Paolo Costantino1 and Maura Cardarelli3
1Dip. Genetica e Biologia molecolare, University of Roma 'La Sapienza', Italy
2Ist. Orticoltura e Coltura Industriale, CNR Via S. Loja 85050 Tito Scalo (Potenza), Italy
3Centro Acidi Nucleici c/o Dip. Genetica e Biologia molecolare, University of Roma 'La Sapienza', Italy
Plant species genetically capable of accumulating toxic metals (hyperaccumulators) offer great opportunity for phytoremediation of contaminated soils. Overexpressing plant genes involved in hyperaccumulation can confer metal tolerance to suitable species.
Our approach is to overexpress these genes in the model species Nicotiana tabacum and evaluate transgenic plants for metal tolerance. In addition, since 'hairy roots' cultures have been demonstrated to be an excellent experimental system to study the absorption of compounds through roots, we used tobacco transformed with a construct containing the gene rolB of Agrobacterium rhizogenes. rolB expression induces an expansion of the root system increasing the absorbing surfaces of roots. Normal and rolB-transformed tobacco seedlings were exposed to different CdSO4 and showed no difference in their resistance to the heavy metal.
Phytochelatins (PC) are peptides overproduced when plants are exposed to heavy metals and play a key role in sequestering them. We isolated the c-DNA that encodes PC synthase from Arabidopsis thaliana and cloned the coding region into the plant binary vector pCambia1302 containing the CaMV35S promoter. rolB-tobacco plants are currently being transformed with this construct. Concomitantly the c-DNA is being used as a probe to evaluate the expression of PC synthase in tobacco seedlings grown in the presence or absence of heavy metals.
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