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Plant Physiol, August 2001, Vol. 126, pp. 1519-1526 Enhanced Copper Tolerance in Silene vulgaris (Moench) Garcke Populations from Copper Mines Is Associated with Increased Transcript Levels of a 2b-Type Metallothionein Gene1Department of Ecology and Ecotoxicology of Plants, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (N.A.L.M.v.H., H.W.J.H., K.F.B., H.S., J.A.C.V., W.H.O.E.); and Department of Biochemistry, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland (V.H.H., S.O.K., A.I.T.)
Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited 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 possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F3 plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.
The Bladder Campion, Silene
vulgaris (Moench) Garcke, has evolved strongly heavy
metal-tolerant populations at sites with high heavy metal
concentrations in the soil (Ernst, 1974 Metallothioneins (MTs) might be involved in copper tolerance. MTs are
low molecular weight, Cys-rich cytoplasmic metal-binding proteins that
could protect cells against the toxic effects of copper by chelating
this heavy metal. Genes encoding MTs occur in animals, higher plants,
eukaryotic micro-organisms, and in some prokaryotes. There are
different MT families, subfamilies, subgroups, and isoforms. A review
of MTs in plants and their characteristics is given by Rauser (1999) The functions of MTs in plants are still unclear (Rauser, 1999 This study was performed to isolate and characterize MT genes from S. vulgaris, and to investigate their role in copper tolerance in different populations of this species. The latter was done by heterologous expression in hypersensitive yeast, as well as by analyzing the co-segregation of MT expression and copper tolerance in segregating families of crosses between copper-sensitive and -tolerant plants.
MT Sequences A cDNA library was prepared from leaves of untreated
copper-tolerant plants (population Imsbach). Of the Arabidopsis-based MT probes tested (MT1a, MT2a, MT2b,
MT3), only MT2b yielded hybridizing plaques. The
corresponding S. vulgaris cDNA (SvMT2b;
GenBank accession no. AF101825) showed a high amino acid
sequence identity with Arabidopsis MT2b (Zhou and Goldsbrough, 1995
Yeast Complementation A copper-sensitive yeast, DBY746, transformed with SvMT2b-pAJ401 was able to grow at 5 mM CuSO4, whereas the untransformed DBY746 mutant grew up to 1 mM copper. Copper tolerance of the copper-sensitive yeast was thus approximately 5-fold increased. The MT-deficient copper-sensitive yeast cup1 mutant DM771-6C transformed with SvMT2b grew at 1 mM CuSO4, but the untransformed mutant did not grow at 0.5 mM copper. A cadmium-sensitive yeast, JWY53 (vacuolar membrane ABC transporter mutant ycf1), transformed with SvMT2b-pAJ401 was able to grow at 0.1 mM CdSO4, whereas the original yeast mutant with or without pAJ401 plasmid grew up to 0.01 mM CdSO4 (Fig. 2).
SvMT2b mRNA Expression Analysis in Parental Ecotypes Both northern-blot hybridization and quantitative RT-PCR showed much higher levels of SvMT2b mRNA in the roots (Fig. 3) and leaves (data not shown) of the plants from the copper-tolerant populations Imsbach and Marsberg than in those from three copper-sensitive populations Amsterdam (Fig. 3), Wijlre and Gaschurn (data not shown). SvMT2b expression was largely unaffected by copper treatment (Fig. 3) and the level of SvMT2b expression was higher in leaves than in roots (data not shown).
Southern-blot analysis showed considerable differences in band intensities between the populations (Fig. 4B), whereas DNA loading was similar for all the samples (Fig. 4A). Digestion of leaf DNA with MboI and hybridization with SvMT2b probe generated a faint band at 1.43 kb for Amsterdam and a very intense band at 1.37 kb for Imsbach and Marsberg. Two smaller faint bands were also detected for Imsbach DNA.
SvMT2b mRNA Expression Analysis in Interecotypic Crosses Two pair crosses were made between plants from the
highly copper-tolerant population Imsbach (EC100 > 400 µM) and the copper-sensitive population Amsterdam
(EC100 < 12 µM). Thirty
F1 plants were pair-crossed to produce
F2 seed. These seeds were pooled and
approximately 500 F2 seedlings were screened for
copper tolerance (see "Materials and Methods"), using
the lowest copper concentration that completely stopped root growth as
a tolerance measure (Schat and Ten Bookum, 1992b The expression levels of the SvMT2b gene in copper-sensitive and copper-tolerant F3 plants were determined to investigate the role of this gene in copper tolerance in S. vulgaris. It seemed that the copper-sensitive F3 plants (1-9) varied discretely in SvMT2b expression (Fig. 5A). The plants from the families 3, 4, and 8 had a low expression level comparable with that of Amsterdam plants. The copper tolerant F3 plants (10-18) showed the same variation in SvMT2b mRNA levels. The plants from the families 10 and 17 showed low expression.
There was a perfect correlation between high SvMt2b expression, as established with quantitative RT-PCR, and the presence of at least one Imsbach allele, as established by allele-specific PCR. Imsbach allele-specific primers did not produce clear bands for the plants of the families with low SvMT2b expression, i.e. 3, 4, 8, 10, and 17, showing that these were homozygous for the Amsterdam allele (Fig. 5B). Only plants from the tolerant families 12 and 16 were homozygous for the Imsbach allele (Fig. 5C). Enhanced SvMT2b expression was more or less evenly distributed over the sensitive and the tolerant F3 selection lines, implying that this gene does not act as a primary determinant in copper tolerance. The possibility remains that it would act as a hypostatic enhancer of the level of tolerance in tolerant plants, however. This was investigated by genotyping less tolerant (EC100 < 100 µM CuSO 4) and more tolerant (EC100 > 150 µM CuSO 4) plants selected from tolerant F4 families that segregated for SvMT2b expression. These F4 families were produced by paircrossing eight tolerant F3 plants with different genotypes for SvMT2b (four crosses between a heterozygote and an Amsterdam-type homozygote, and one heterozygote × heterozygote cross) but equal levels of tolerance. The resulting five F4 families were screened for tolerance (25 plants per family) and the five least tolerant, as well as the five most tolerant plants of each of the families were genotyped for SvMT2b (see below). High-level tolerance appeared to be significantly positively associated with the possession of the highly expressed Imsbach allele (G-test with Yates' correction: G = 21.7; P < 0.001). Plants lacking the Imsbach allele were over-represented among the less tolerant plants (expected 43%, observed 90% [n = 20]), whereas plants possessing the Imsbach allele were over-represented among the more tolerant ones (expected 57%, observed 85% [n = 20]) (Fig. 6).
Pair crosses were also made between highly copper-tolerant plants from the population Imsbach and the moderately copper-tolerant plants from the population Marsberg. Three F1 plants derived from different crosses were selfed, and the F2 families were screened for tolerance. Six of the least tolerant (EC100: 50-100 µM CuSO4) and six of the most tolerant (EC100 > 250 µM CuSO4) F2 seedlings of each F2 family were genotyped for SvMT2b. There was no significant co-segregation of allele origin with copper tolerance in these crosses. Sixteen of 18 plants tested, both from the low-tolerance and high-tolerance selected lines, possessed one or two Imsbach alleles, which strongly suggests that the differential tolerance of Imsbach and Marsberg plants is unrelated to the difference in the primary structure of the predicted SvMT2b proteins.
The SvMT2b gene was classified as type 2 within the
MT-II class due to the presence of Cys-Cys and Cys-×-×-Cys motifs in
the N-terminal domain (Robinson et al., 1993 The Southern blot showed big differences in band intensities between
Amsterdam, Marsberg, and Imsbach (Fig. 3b). Recent results have
revealed that SvMT2b is composed of two exons and a long intron, inserted after bp 69 of the coding sequence (A. Tervahauta, unpublished data). The SvMT2b coding sequence contains one
MboI restriction site located in exon I. The probe that we
used was specific to exon II, implying that the number of
SvMT2b containing restriction fragments should be equal to
the number of copies of this gene in the genome. The probe, which
completely matched exon II from Imsbach plants, contained 5- and 4-bp
mismatches with exon II from Amsterdam and Marsberg plants,
respectively, which is far from sufficient to produce the band
intensity differences under the stringency conditions applied. Thus, it
seems that the intense bands in Imsbach and Marsberg must have resulted
from the presence of a number of identical SvMT2b containing
repeats. The size of these repeats is unknown, but may be longer than
1.37 kb, because the intron, which has not been completely sequenced, contains at least two MboI sites (A. Tervahauta, unpublished
data). Further analysis of the repeat structure by long PCR and reverse PCR has been unsuccessful thus far. Tandem repeat of SvMT2b
seems to be likely however, and might account for the overexpression in
the tolerant plants. To our knowledge, no other plant MT tandem repeat
has been described so far. However, a tandem repeat of the yeast MT
gene, CUP1, has been found in cadmium-resistant (Tohoyama et
al., 1996 High SvMT2b mRNA accumulation in plants from the
Amsterdam × Imsbach F3 crosses was strictly
dependent on the presence of at least one Imsbach allele.
Low-expression F3 plants were all homozygous for
the Amsterdam allele. This implies that high expression could be
conferred by a dominant cis-acting regulatory component or, possibly, a
closely linked trans-acting component. Enhanced SvMT2b
expression as such is not sufficient to produce increased copper
tolerance, relative to the sensitive parent population, as indicated by
the presence of high-expression plants among the non-segregating
copper-sensitive F3 families. However, a strict co-segregation between high-level copper tolerance and
SvMT2b overexpression did occur in tolerant
F4 families (Fig. 5). Thus, overexpression of
SvMT2b can confer additional copper tolerance, although only
in combination with components of the genetic background of a copper
tolerant plant, suggesting that SvMT2b acts as a hypostatic enhancer, rather than as a primary tolerance gene. An important role
for hypostatic enhancers in copper tolerance has also been demonstrated
in Mimulus guttatus (Macnair, 1983 There are differences in the predicted amino acid sequences of the
SvMT2b protein between the S. vulgaris populations. This could result in a change in the three dimensional structure of the
protein (Keeton et al., 1993 Overexpression of SvMT2b restored cadmium and copper
tolerance in hypersensitive yeast, suggesting that the SvMT2b protein increased the cellular sequestration capacity, probably through binding
the metals. We were unable to assess the cellular SvMT2b protein levels
in S. vulgaris, probably because of their sensitivity to
oxidation. However, the highly significant cosegregation of enhanced
SvMT2b expression with high tolerance in tolerant
F4 families indicates that the mRNAs were indeed
translated into functional proteins. It is very difficult to detect
this type of MT proteins, therefore not much is known about
post-transcriptional processing of MTs. However, Murphy et al. (1997) SvMT2b overexpression in both the Imsbach and Marsberg
populations must have resulted from independent evolution. Gene flow between these populations is very unlikely (Schat et al., 1996 In conclusion, the copper-tolerant populations showed a constitutively higher SvMT2b expression, which could result from gene amplification. This overexpression does not produce copper tolerance by itself but merely increases the level of tolerance produced by one or more epistatic primary tolerance genes. Further studies of copper tolerance genes and their interactions are necessary to get a better understanding of the different mechanisms involved in copper tolerance in S. vulgaris.
Plant Materials Seeds of Silene vulgaris plants were collected
from the copper mines near Imsbach and Marsberg (Germany) and from the
botanical garden of the Vrije Universiteit of Amsterdam (The
Netherlands). Characteristics of these sites and of the local S.
vulgaris populations have been given in Schat and Ten Bookum
(1992a) Tolerance Testing Seed germination, hydroponic preculture, nutrient and test
solution compositions, and growth chamber conditions were exactly as in
Schat et al. (1996) cDNA Library Construction and Screening A Characterization of SvMT2b Expression Patterns Plant Culture The plants were grown hydroponically for 2 weeks before DNA and/or RNA extraction. Some of the plants were exposed to 50 µM CuSO4 for 24 or 48 h before harvest. At harvest, roots were shortly rinsed with distilled water and the whole root system or/and a mature leaf was cut off, immediately frozen in liquid nitrogen, and stored at 80°C until extraction.
DNA/RNA Isolation DNA isolation was performed according Doyle and Doyle (1990) .
Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen).
cDNA Preparation Single-stranded cDNA was prepared from 2 µg of RNA, 20 pmol of RACE dT primer, 0.25 mM deoxynucleoside triphosphates (Life Technologies/Gibco-BRL, Cleveland), 200 units of M-MLV reverse transcriptase (Life Technologies/Gibco-BRL), and 1 × RT first strand buffer (Life Technologies/Gibco-BRL), in a total volume of 20 µL.Sequencing of SvMT2b Alleles The primers 5'-TTTCAGTAATTTAATCAGCG-3' and 5'-GCTTGTTTTACCCTGTTGAG-3', based on the non-coding regions of a MT2b-like cDNA (SvMT2b) found in the library (see above), were used to amplify and sequence the coding regions of the corresponding cDNAs of plants from the populations Amsterdam and Marsberg. PCR was performed using 2 µL of cDNA, 20 pmol of each primer, 1 mM deoxynucleoside triphosphates (0.25 m each) (Life Technologies/Gibco-BRL), 1 unit of Taq polymerase (MRC Holland), and 1 × Taq buffer (MRC Holland), adjusted to a total volume of 25 µL with sterilized water. An annealing temperature of 55°C and 34 cycles were used. PCR products of the expected lengths were sequenced. Sequencing was performed using the Terminator Cycle Sequencing Core Kit (Perkin-Elmer Applied Biosystems, Foster City, CA).Allele-Specific PCR The primers used for the allele-specific PCR were 5'-GAAAATGTCGTGCTGTAATGGA-3' and 5'-GTTCATTTGCAAGTGCAAGGG-3' for the Imsbach allele and 5'-GAAAATGTCGTGCTGTAATGGT-3' and 5'-GCGTCGAAGTATGAGGCCTT-3' for the Amsterdam allele, giving SvMT2b PCR products of 243 and 159 bp, respectively. The allele-specific PCR was performed using 2 µL of cDNA or 100 ng of total genomic DNA, 20 pmol of each primer, 1 mM deoxynucleoside triphosphates (0.25 mM each) (Life Technologies/Gibco-BRL, Cleveland), 1 unit of Taq polymerase (MRC Holland), and 1 × Taq buffer (MRC Holland), adjusted to a total volume of 25 µL with sterilized water, in a PCR program with 34 cycles and an annealing temperature of 69°C (2 min 94°C; 34 cycles of [30 s 94°C, 45 s 69°C, 1 min 30 s 72°C]; 5 min 72°C; storage at 4°C) (Biometra Tgradient). Eight microliters of each PCR product was run in an agarose gel with a 250-bp DNA mass ladder (MRC Holland) and photographed in UV-light.Quantitative RT-PCR The primers used for the quantitative RT-PCR were 5'- GAAAATGTCGTGCTGTAATGG-3' and 5'-AAGGGTTGCACTGGCAGTTG-3', based on a part of the SvMT2b cDNA that was identical in plants from Amsterdam, Marsberg, and Imsbach. The house-keeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a positive internal control. The primers 5'-TGGGATCCTCACTGACAAGGACAAGGCT-3' and 5'-TGAATTCCCCCATTCGTTGTCGTACC-3', designed on the basis of published sequences of GAPDH genes from other plant species (GenBank Accession no. X75597; Niu et al., 1994; GenBank Accession no.
X07156; Brinkmann et al., 1987), were used to identify S.
vulgaris GAPDH. PCR and sequencing of a part of the
GAPDH gene, using cDNA of plants from Imsbach as a
template, was performed as described in "Sequencing of SvMT2b alleles" (see above). The S. vulgaris GAPDH primers
used for the quantitative RT/PCR were 5'-GTTATCATCTCAGCTCCTAG-3' and
5'-GCAACACCTTTCCAACAGCCT-3'. Quantitative PCR was performed using 4 µL of cDNA, 20 pmol of each primer, 1 mM deoxynucleoside
triphosphates (0.25 mM each) (Life Technologies/Gibco-BRL),
1 unit of Taq polymerase (MRC Holland), and 1 × Taq buffer (MRC Holland), adjusted to a total
volume of 25 µL with sterilized water. A PCR program of
15 cycles and an annealing temperature of 55°C was used (2 min
94°C; 15 cycles of [30 s 94°C, 45 s 55°C, 1 min 30 s
72°C]; 5 min 72°C; storage at 4°C) (Biometra Tgradient). This
gives SvMT2b and GAPDH PCR products of
227 and 313 bp, respectively. Twelve microliters of each PCR product
was electrophoresed in an agarose gel and photographed in UV light.
Northern-Blot Analysis Eight micrograms of total RNA isolated from roots of untreated and copper-treated (50 µM CuSO4 for 0, 24, and 48 h) plants from the populations Amsterdam and Imsbach were used. Preparations of samples, formaldehyde agarose gel electrophoresis, and blotting on nylon membrane was performed according to Sambrook et al. (1989). The primers used to make
the radioactive cDNA probe were 5'- GAAAATGTCGTGCTGTAATGG-3' and
5'-CATTTGCAAGTGCAAGGGT-3' and Imsbach cDNA was used as a template, resulting in a probe of 240 bp. Labeling of the radioactive probe (isotope 32P), overnight hybridization at
65°C and washings (brief rinsing at 65°C in 2× SSC and washing for
10 min in 0.5× SSC/0.1% [w/v] SDS at 65°C) were performed
according to Church and Gilbert (1984). The blot was scanned with a
phosphor imager.
Southern-Blot Analysis Six micrograms of total DNA, isolated from leaves of plants from the populations Amsterdam, Marsberg, and Imsbach, was digested overnight at 37°C with the restriction enzyme MboI and electrophoresed overnight on a 0.7% agarose gel in 0.5 × TBE buffer with a DIG-labeled DNA marker II (Boehringer Mannheim). DNA was blotted on a Nylon membrane (Boehringer Mannheim). The primers used to make the cDNA probe were 5'-CAAGATGTTCCCTGAGTTT-3' and 5'-CATTTGCAAGTGCAAGGGT-3' and Imsbach cDNA was used as a template, resulting in a probe of 168-bp (part of the gene without an intron). DIG labeling of the probe, hybridization, and washings were performed as described in the Boehringer Mannheim manual (GmbH DIG system; 1995). Hybridization occurred overnight at 42°C. The blots were washed twice in 2× SSC/0.1% (w/v) SDS at room temperature for 15 min and two times more in 0.2× SSC/0.1% (w/v) SDS at 68°C for 15 min. The blots were exposed to a chemiluminescence film (Hyper film ECL, Amersham Life Science). DNA loading was checked by ethidium bromide staining.Complementation of Copper-Sensitive and Cadmium-Sensitive Yeast Mutants SvMT2b was amplified with PCR from a
The plasmid pAJ401 was kindly provided by Dr. Anu Saloheimo (Biotechnology and Food Research, VTT, Finland). The JWY53 yeast strain was supplied by Professor W. Scott Moye-Rowley (The University of Iowa College of Medicine), Professor Peter Goldsbrough (Purdue University) provided the Arabidopsis MT-plasmids. The authors would also like to thank Gregory Koningstein (Department of Molecular Microbiology, Faculty Biology, Vrije Universiteit, Amsterdam) for assisting with the sequencing of the SvMT2b cDNAs.
Received December 22, 2000; returned for revision February 14, 2001; accepted April 19, 2001. 1 This work was supported by the European Community (Environment Research Program: Environment and Climate, contract no. ENV 4-CT95-0083 [Phytorehab]).
* Corresponding author; e-mail hschat{at}bio.vu.nl; fax 31-20-4447123.
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ABSTRACT
INTRODUCTION
ycf1) (upper) and DBY746 (lower) growing
on PDA plates supplemented with cadmium or copper as sulfates: 1, untransformed; 2 and 4, transformed with empty vector (pAJ401); and 3, transformed with SvMT2b.
12 µM) and 12 copper-tolerant seedlings
(EC100 
100 µM) were selected and
pair-crossed (sensitive × sensitive and tolerant × tolerant) to produce nine sensitive and nine tolerant F3 families. Twenty-four plants of each of these
families were screened for tolerance. The copper-sensitive
F3 families appeared to be devoid of any
copper-tolerant individuals. The tolerant F3
families showed a degree of segregation (EC100
values between 50 and 400 µM CuSO4)
but were devoid of any copper-sensitive individuals. The average
tolerance levels in these families (EC100 between 100 and 200 µM) were consistently lower than that of the
tolerant parent population Imsbach (EC100
approximately 500 µM).
gt11 cDNA library was prepared from leaves of
untreated copper-tolerant plants (population Imsbach). Total RNA was
isolated according to the guanidine hydrochloride method (Logemann et
al., 1987
