SYTTY
PHYTOREMEDIATION OF METAL POLLUTED SOILS: MECHANISMS AND MANIPULATION OF METAL TOLERANCE AND HYPERACCUMULATION IN PLANTS
Project leader: Sirpa Kärenlampi, University of Kuopio,
Department of Biochemistry P.O.Box 1627, FIN-70211 Kuopio, Finland,
tel. +358-17-163069, e-mail: Sirpa.Karenlampi@uku.fi
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Reseachers:
Professor Sirpa Kärenlampi, University of Kuopio, Department of
Biochemistry, tel. +358-17-163069, e-mail: Sirpa.Karenlampi@uku.fi
Arja Tervahauta, Ph. D., Department of Biochemistry, University of
Kuopio, tel. +358-17-163063, e-mail : Arja.Tervahauta@uku.fi
Viivi Hassinen, M. Sc., Department of Biochemistry, University of Kuopio,
tel. +358-17-163063, and North Savo Regional Environment Centre
e-mail: Viivi.Hassinen@uku.fi
Kaisa Koistinen, M. Sc., Department of Biochemistry, University of
Kuopio, tel. +358-17-163058, e-mail: Kaisa.Koistinen@uku.fi
Sirpa Keinänen, M. Sc., Department of Biochemistry, University
of Kuopio, tel. +358-17-163058, e-mail: Sirpa.Keinanen@uku.fi
Harri Kokko, M. Sc., Department of Biochemistry, University of Kuopio,
tel. +358-17-163200, e-mail: Harri.Kokko@uku.fi
Kristina Servomaa, Ph. D., North Savo Regional Environment Centre,
tel. +358-17-7884900, e-mail: Kristina.Servomaa@vyh.fi
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 213329
/ 459163
Person-months of work funded by SYTTY / Total person-months of work:
74 / 214,5
KEY WORDS: Phytoremediation, soil, plants, metal tolerance, molecular
biology
EXTENDED ABSTRACT
1 Introduction
Heavy metals are natural elements cycling at low levels in bio-, geo-, atmos- and hydrospheric systems. At optimal levels, metals like Cu, Zn, Mn, Fe, Ni and Mo are essential to biological systems. At increased levels, both essential and non-essential metals (Cd, Pb, Hg) metals are toxic. Local metal increases are caused by human activities: smelting, mining, processing, agricultural and waste disposal technologies. Metal concentrations in soils are increasing, leading to potential increases in leaching to water, uptake by plants and intake by human population. This is an increasing risk for human health and the environment. Contamination also affects the growth and survival of plants and microbes. Some species have adapted to increased metal concentrations, and have developed a heritable tolerance to heavy metals. Some small and slowly growing metal hyperaccumulator plants survive even in metal-rich soils; these plants absorb heavy metals effectively into the roots, and transfer them to the above-ground parts. The mechanisms of metal tolerance in plants are poorly known, as are also the mechanisms of metal hyperaccumulation. By studying the genes that are regulated by metal exposure as well as genes that are linked to metal tolerance we hope to find out new aspects of these mechanisms. By genetic studies we also hope to find ways to influence the uptake and distribution of metals in the plant. The information can be used to produce plants optimized for purification of agricultural fields and remediation of soils around industrial emission sites and, by that way, to improve the quality of the environment. Tolerant plants can be used for revegetation of metal contaminated land, which also helps immobilizing the remaining metals in the soil. Improvement of metal exclusion from edible plants may advance human nutrition and thus human health.
2 Methods
Isolation of putative metal tolerance genes from Silene vulgaris and
complementation in yeasts
A lambda gt11 cDNA library was prepared from leaves of a metal-tolerant
S. vulgaris (Imsbach population). Library has been screened with several
DIG-labeled microbial probes linked to metal tolerance as well as with
metallothionein genes (MT1a, MT2b, MT3) and paa1 gene encoding a Cd-binding
ATPase from Arabidopsis thaliana. Also an oligonucleotide probe was made
for the metal-binding domain (MBD) of Cd-ATPase. MT from Silene vulgaris
was cloned into pAJ401 E. coli - yeast shuttle vector. Recombinants were
first introduced into E. coli and selected for ampicillin resistance and
the presence of MT was confirmed with PCR. Plasmid DNA was introduced into
Cd-sensitive (JWY53) and Cu-sensitive yeasts (DBY746). The first transformant
selection in yeast was made for URA auxotrophy, and then by increased metal
tolerance. Using RT-PCR the MT expression was studied in metal tolerant
and sensitive Silene plants by Ms Nathalie van Hoof in the Free University
of Amsterdam.
Isolation and sequencing of 5 prime upstream fragment of Silene MT
gene
To isolate and sequence the 5 prime upstream regulatory region of Silene MT gene, Extender PCR was used for walking into previously uncloned regions of genomic DNA. A single-stranded oligonucleotide adaptor was ligated to restriction enzyme-digested genomic DNA and the blocking of non-specific replication of the adaptor-complementary strand was done by incorporation of a dideoxynucleotide. Unknown adaptor primer-ligated fragment next to known MT sequence was then amplified by using adaptor primer and MT-specific primer and sequenced.
Isolation of metal tolerance and hyperaccumulator genes from Thlaspi
caerulescens
Exposures at four different Zn concentrations in hydroponic culture
were made for five different populations of T. caerulescens, which differ
in their metal uptake, translocation and tolerance. In order to find differentially
expressed genes in different populations and Zn exposures, a DDRT-PCR analysis
was made. The expression of the found gene fragments was studied using
Reverse Northern and Northern blotting -methods. To further elucidate the
role of the genes found, a new hydroponic culture was done. Five T. caerulescens
populations were exposed to high Cd or Zn or Zn deprivation, and the samples
were used in Northern blot experiments.
Isolation of metal tolerance genes from Betula pendula.
Cu-exposures were made in hydroponic culture at three different metal
concentrations. Two different birch clones were studied, a Cu-sensitive
(Wales 002) and a Cu-tolerant (Wales 008). Leaf and root samples were taken
for RNA-isolation. cDNA subtraction library have been made by CLONTECH
PCR-Select cDNA Subtraction Kit for finding differentially expressed genes
those genes expressed in one mRNA population, but reduced or absent in
other. Differentially expressed genes were studied by reverse Northern
dot blot hybridization and most promising genes were studied by Northern
blot analysis.
Identifying of copper-induced proteins in birch
The response of a Cu- and Zn-tolerant birch (Betula pendula) clone
to copper stress was investigated. Two-dimensional electrophoresis was
used to compare the differences in protein expression pattern between Cu-exposed
and control plants in roots and leaves. Proteins were identified using
on-line high-performance liquid chromatography - electrospray ionisation
- ion trap mass spectrometry.
Analysis of PR-10c induction in birch
Antibody against the C-terminal peptide of PR-10c was made and used
in Western blot studies of leaves and roots of Cu-, Zn- and Cd-exposed
birches. A His-Tag fusion protein of PR-10c was produced in E. coli for
a positive control in Western blot analysis. The PR-10c gene was introduced
in metal sensitive yeast mutants.
Analysis of function and post-translational modifications of PR-10c
protein
HPLC-ESI and MALDI-TOF mass spectrometry were used for analysis of
post-translational modifications of PR-10c protein. Reduced and S-glutathiolated
forms of PR-10c-His fusion protein were used for RNase activity gel assay.
Nuclease activity was studied by incubating both forms of PR-10c-His fusion
protein with RNA and different DNA substrates.
Transformation of tobacco
The bacterial metal resistance genes cadA and pcoA were cloned in the
binary vector pBI121 to be expressed as transcriptional fusions with reporter
gene gus. The cot1 gene from Saccharomyces cerevisiae was cloned in pBI121
vector so that gus would not be in-frame. The constructs were transferred
into Agrobacterium tumefaciens LBA4404 cells and selected for kanamycin
resistance. Leaf pieces from greenhouse grown tobacco (SR1, Little Havanna)
were inoculated with recombinant Agrobacterium, and the shoots were selected
with kanamycin. The transformants were analysed with PCR using gene-specific
primers. The positive plants were further studied by Southern blot hybridization.
The expression at mRNA level was studied by Northern blot hybridization
and with RT-PCR. To study expression of metal tolerance genes at protein
level, antibodies were raised against synthetic peptides and fusion proteins
in rabbits and mice. His-tagged fusion proteins were produced for positive
controls in Western blot and as antigens for immunization. Total and membrane
proteins from the transgenic plants were studied with specific antibodies
in Western blot. The F1 plants were exposed to metals in either soil or
hydroponic cultivation. Leaf and root samples were taken for metal analysis
(atomic absorption spectrometer).
Modification of pcoA gene for optimal expression in plants
Due to low gene pcoA gene expression in tobacco, DNA sequence was modified
to change the codon usage for optimized expression in plant. Bacterial
signal sequence was removed and mutations were introduced using long primers
in PCR. Modified gene was cloned in pYES2.1/V5-His-TOPO yeast E. coli
shuttle vector and transferred to metal sensitive yeast mutants (JWY53,
YAW10, DM771-1C) to study the effect on metal tolerance and uptake.
3 Results and Discussion
Isolation of putative metal tolerance genes from S.vulgaris
A lambda gt11 cDNA library of S. vulgaris was screened with several
plant probes. With A. thaliana MT2b probe a Silene metallothionein gene
(Genbank accession number AF101825) was found which shares closest homology
with the metallothionein gene of Mesembryanthemum crystallinum (ice plant).
The library was also screened with microbial probes linked to metal tolerance
but no homologies were found. This may be due to the fact that plants and
microbes use different codons for proteins with similar function and polypeptide
sequence.
Silene MT and heavy metal tolerance
In order to study whether Silene MT gene is related to heavy metal
tolerance, it was introduced into metal sensitive yeast mutants. The result
was an increase in metal tolerance as shown in Table 1.
Table 1. Growth of yeast mutants and MT-transformed yeasts in metal-containing
medium. The highest concentrations where the yeasts grew are shown.
| Yeast mutant | Untransformed yeast | MT-transformed yeast |
| DBY746 (CuS) | 1 mM Cu | 5 mM Cu |
| DM771-6C (CuS, ?cup1) | 0,5 mM Cu | 1 mM Cu |
| JWY53 (CdS, ?ycf1) | 0,01 mM Cd | 0,1 mM Cd |
Cd tolerance of the Cd-sensitive yeast JWY53 (vacuolar membrane ABC transporter mutant ycf1) was thus increased by about 10-fold by phenotypic complementation. The sensitivity of the yeast mutant was apparently due to the lack of transport of Cd to the vacuole; the plant MT gene product detoxified cytosolic Cd by binding into it. Cu tolerance of the Cu-sensitive yeast DBY746 was increased by about 4-fold. The Cu-sensitive yeast mutant DM771-6C (mutation in cup1 locus, i.e. yeast MT) transformed with Silene MT grew at 1 mM CuSO4 but the untransformed yeast did not grow at 0.5 mM Cu. The Silene MT gene thus caused a genotypic complementation of the yeast mutant. Ms Nathalie van Hoof in the Free University of Amsterdam studied MT expression in metal tolerant and sensitive Silene plants by RT-PCR, and found that it was higher in tolerant plants. According to her studies MT is not the main tolerance gene but it cosegregates with tolerance, being probably a modifier gene.
5 prime upstream fragment of Silene MT gene
A 283 bp fragment from the 5 prime upstream region was isolated from
tolerant and sensitive plants and sequenced in order to detect differences
in their promoter sequences. Two nucleotide differences were found but
the importance of the finding is not yet clear.
Isolation of metal tolerance and hyperaccumulator genes from T. caerulescens
Over 35 differentially expressed gene fragments were found from two
populations of T. caerulescens when Zn exposed and non-exposed leaf samples
were compared. The fragments have been sequenced. Some of the fragments
showed homology to identified genes or genes known to be involved in metal
binding and transport in Arabidopsis thaliana; the rest of the fragments
have only weak homology or they represent unknown genes. The search of
the full-length genes from a Thlaspi cDNA-library has started. The expression
of more than 25 fragments has been studied. Most of the genes represented
by the fragments are regulated by metals and the populations have differences
in expression profile. Further studies are needed to understand the role
and also the regulation of the genes in plant metal detoxification.
Metal- induced genes from birch
The response of a Cu- and Zn-tolerant birch (B. pendula) clone to Cu
stress was previously investigated by 2D-electrophoresis, the most apparent
quantitative difference being the increased amount of a 17 kDa protein,
identified as PR-10c (Bet v 1-Sc3), in both roots and leaves. Expression
of PR-10c was studied by Western blot in Cu, Cd and Zn-exposed roots and
leaves, and induction was found with all metals in both leaves and roots.
When transferred into metal-sensitive yeast mutants, PR-10c did not improve
metal tolerance. Mass spectrometric analysis revealed that PR-10c protein
is post-translationally modified by glutathione in vivo in the roots of
zinc-exposed birch. The possible nuclease activity of PR-10c was analysed
with S-glutathiolated and reduced fractions of PR-10c-His fusion protein.
Both forms possessed RNase activity, which is capable of digesting different
RNA substrates.
Production of transgenic tobacco plants
Microbial metal tolerance genes were transferred into tobacco to improve
heavy metal tolerance and/or to increase metal uptake. The pcoA gene seems
so far the most promising system in increasing metal uptake. None of the
cadA transformants showed an altered uptake of metals into leaves compared
to SR1 parent tobacco. Tobacco transformants for cot1 gene were also studied
for metal uptake, but no increase in their metal content was found.
Modification of pcoA gene for optimal expression in plants
Signal sequence was removed and several nucleotide changes were made
in the pcoA sequence. In the proces, few unintended point mutations were
introduced. The modified gene was transferred in metal-sensitive yeasts,
but did not increase metal tolerance or uptake.
4 Conclusions
Silene metallothionein confers metal tolerance for metal-sensitive yeasts,
and is probably a metal tolerance modifier gene in Silene.
PR-10c protein from Cu-exposed birch is not directly related to metal
tolerance, but is a putative biomarker for (oxidative) stress.
PR-10c protein is post-translationally modified by glutathione in vivo
in the roots of zinc-exposed birch, and both S-glutathiolated and reduced
fractions of PR-10c possess RNase activity.
Metal uptake of tobacco may be increased with some microbial heavy
metal tolerance genes.
Number of known and unknown metal-related/metal responsive genes have
been isolated from plants and their characterization has been initiated.