Gene expression induced by copper stress in the diatom Thalassiosira pseudonana

Abstract

Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by approximately 500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.

FIG. 1.

Cue genes located within an inverted repeat on genomic scaffold 73. (A) Gene induction after 1 and 24 h of copper exposure monitored by measuring mRNA levels with RT-PCR. mRNA concentrations are means normalized to control cells that were not treated with Cu. Error bars indicate standard deviations from two technical RT-PCR replicates. Note that the y axis is a logarithmic scale. (B) Schematic diagram of the inverted repeat region, indicated by gray bars. Cue gene designations and orientations are shown. The figure is shown to scale except for the intervening region between the inverted repeats, which is indicated by a double bar.

FIG. 2.

Amino acid sequence comparisons of predicted Cue proteins located within an inverted repeat on genomic scaffold 73. Alignments were performed with CLUSTAL W (1.81) (54). Sequences are identified on the left, and amino acid residue numbers are indicated on the right. Dashes in sequences were introduced to facilitate maximum alignment. Below the sequences, asterisks denote amino acid residues conserved in all sequences and dots indicate conservative replacements. Signal sequences were predicted with SignalP 3.0 (6) and are enclosed within light gray boxes. (A) Alignment of Cue1, Cue2, Cue6, Cue7. The repeated domain in Cue6 and Cue7 is encompassed within unshaded boxes. PredictNLS (10) identified a potential NLS in Cue1 and Cue2, denoted by dark gray boxes. (B) Alignment of Cue18 and Cue19.

FIG. 3.

Changes in mRNA concentrations corresponding to identified genes, other than those presented in Fig. 1, measured by RT-PCR after 1 and 24 h of copper exposure. mRNA concentrations were normalized to control cells and are expressed in relative units. Note that the y axis is a logarithmic scale. RT-PCR results for p150, a gene whose induction by 24 h of copper exposure has already been described (11), as well as results for a similar gene within the genome, designated “p150-like,” are shown as examples of genes known to be induced by copper stress. Nitrate reductase (NR) was identified in a subtracted cDNA library enriched in genes down-regulated by 1 h of copper exposure and is included as an example of a down-regulated gene.

FIG. 4.

Effects of H₂O₂ exposure on T. pseudonana. (A) Growth of T. pseudonana exposed to various concentrations of H₂O₂. Cell growth was followed by measurement of chlorophyll fluorescence, and results are presented in relative fluorescence units (RFU). Results shown are of a representative growth experiment. Cells grown in batch culture were aliquoted into different treatment conditions on day 0. Error bars represent standard deviations for replicate cultures and are contained within the corresponding symbol when not visible. (B) Gene expression changes monitored by measuring mRNA levels with RT-PCR after 1 and 24 h of exposure to 0.1 mM H₂O₂. mRNA concentrations were normalized to control cells and are expressed in relative units. Note that the y axis is a logarithmic scale. mRNA levels from copper-exposed cells are also presented for comparison and are represented as dashed lines and bars.

FIG. 5.

Effects of Cu and Cd exposure on T. pseudonana. Growth of T. pseudonana exposed to various concentrations of Cu (A) and Cd (B) is shown. Cell growth was followed by measurement of chlorophyll fluorescence, and results are presented in relative fluorescence units (RFU). Results shown are of representative growth experiments. Cells grown in batch culture were aliquoted into various treatment conditions on day 0. Error bars represent standard deviations for replicate cultures and are contained within the corresponding symbol when not visible. Expression changes of a subset of Cue genes were monitored by measuring mRNA levels with RT-PCR after cells had been exposed for 1 h to either Cu (C) or Cd (D) at the concentrations indicated in the keys at right. Note that the y axes are logarithmic scales.