Expression of zinc and cadmium responsive genes in leaves of willow (Salix caprea L.) genotypes with different accumulation characteristics

Abstract

Salix caprea is well suited for phytoextraction strategies. In a previous survey we showed that genetically distinct S. caprea plants isolated from metal-polluted and unpolluted sites differed in their zinc (Zn) and cadmium (Cd) tolerance and accumulation abilities. To determine the molecular basis of this difference we examined putative homologues of genes involved in heavy metal responses and identified over 200 new candidates with a suppression subtractive hybridization (SSH) screen. Quantitative expression analyses of 20 genes in leaves revealed that some metallothioneins and cell wall modifying genes were induced irrespective of the genotype's origin and metal uptake capacity while a cysteine biosynthesis gene was expressed constitutively higher in the metallicolous genotype. The third and largest group of genes was only induced in the metallicolous genotype. These data demonstrate that naturally adapted woody non-model species can help to discover potential novel molecular mechanisms for metal accumulation and tolerance.

Fig. 1

Two metallothionein genes are predominantly induced in leaves upon Zn + Cd treatment and thus display category (1) expression pattern. qPCR analysis of genotypes KH21 (full bars) and F20 (striped bars) after 2, 8 and 14 weeks in perlite cultures without (control, gray) and with 5 mg Zn L⁻¹ and 0.5 mg Cd L⁻¹ (Zn + Cd, black). Expression levels of ScMT2B (a) and ScMT3 (b) were normalized to ScACTIN2. Measurements were carried out in triplicates and values represent means ± SE of four biological replicates. Different letters represent significant differences (Student's t-test, p < 0.05).

Fig. 2

The cysteine biosynthesis gene serine-o-actetyl transferase (ScSAT) is constitutively higher expressed in leaves of the metallicolous isolated KH21 and thus displays a category (2) expression pattern. qPCR analysis of genotypes KH21 (full bars) and F20 (striped bars) after 2, 8 and 14 weeks in perlite cultures without (control, gray) and with 5 mg Zn L⁻¹ and 0.5 mg Cd L⁻¹ (Zn + Cd, black). Expression levels of ScSAT were normalized to ScACTIN2. Measurements were carried out in triplicates and values represent means ± SE of four biological replicates. Different letters represent significant differences (Student‘s t-test, p < 0.05).

Fig. 3

Glutathione biosynthesis and transporter genes are only induced after Zn + Cd treatment in leaves of the metallicolous genotype and thus display category (3) expression pattern. qPCR analysis of genotypes KH21 (full bars) and F20 (striped bars) after 2 weeks in perlite cultures without (control, gray) and with 5 mg Zn L⁻¹ and 0.5 mg Cd L⁻¹ (Zn + Cd, black). Expression levels of the glutathione biosynthesis genes, ScOASA1 and ScCAD2, and the transporter genes, ScZIP6 and ScHMA1, were normalized to ScACTIN2. Measurements were carried out in triplicates and values are means ± SE of four biological replicates. Note that ScZIP6 is constitutively higher expressed in F20 in control condition and downregulated to the level of KH21 upon Zn + Cd exposure. Different letters represent significant differences (Student's t-test, p < 0.05).

Fig. 4

qPCR expression analysis in leaves of selected genes identified in the SSH screen. Expression values were normalized to ScACTIN2 and represent means ± SE of six biological replicates after either 8 or 14 weeks of Zn + Cd treatment. Abbreviations of genes correspond to Supplementary Table S3. Significance levels were calculated with Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).