Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene

Abstract

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 F(3) 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 F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Figure 1

Alignment of amino acid sequences of S. vulgaris SvMT2b (population Imsbach; Sv), Arabidopsis MT2b (At), and M. crystallinum MT (Mc).

Figure 2

Expression of SvMT2b in yeast. Yeast strains JWY53 (Δycf1) (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.

Figure 3

SvMT2b expression in roots of copper-sensitive (Am, Amsterdam) and copper-tolerant plants (Im, Imsbach), unexposed (0) or exposed to 50 μm CuSO₄ for 24 and 48 h (respectively, 24 and 48). A, Ethidium bromide-stained agarose gel of the quantitative RT-PCR products. Also included is the internal control, GAPDH. B, Northern-blot analysis. Each lane was loaded with 8 μg of total RNA. Radioactively labeled SvMT2b was used as a probe.

Figure 4

Amplification of SvMT2b sequences in copper-tolerant populations. A, Electrophoresis of the MboI-digested DNA (ethidium bromide-stained gel; each lane was loaded with 6 μg total leaf DNA of a single plant, digested with MboI). Arrows indicate the sizes of the marker fragments (DNA molecular-weight marker II, DIG-labeled [Boehringer Mannheim]; from the top, 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.6 kb, respectively). B, Southern hybridization using a DIG-labeled SvMT2b cDNA probe. Sizes of the fragments were estimated by reference to the sizes of the marker. Am, Amsterdam; Im, Imsbach; Ma, Marsberg; M, Marker.

Figure 5

SvMT2b expression in copper-sensitive (1–9) and copper-tolerant (10–18) F₃ plants. A, Quantitative RT-PCR products of SvMT2b in root RNA (the plants were grown on 0.1 μm CuSO₄; a quantitative RT-PCR of GAPDH was used as an internal control). Allele-specific PCR products of SvMT2b obtained with Imsbach-specific primers (B) and with Amsterdam-specific primers (C). Am, Amsterdam; Im, Imsbach; Ma, Marsberg.

Figure 6

Cosegregation of SvMT2b allele origin and copper tolerance in F₄ lines derived from crosses between copper-tolerant F₃ plants. PCR products of SvMT2b using Imsbach allele-specific primers and leaf DNA from plants of the S. vulgaris populations Amsterdam and Imsbach, and from 12 plants of different F₄ lines (C1, C2, and C3). Low-tolerance plants and high-tolerance plants were selected from crosses between tolerant low-expression homozygotes and tolerant high-expression heterozygotes. M, Marker (250-bp DNA mass ladder, MRC Holland); Am, Amsterdam; Im, Imsbach.