Whole-genome expression analysis reveals a role for death-related genes in stress acclimation of the diatom Thalassiosira pseudonana.

Low iron (Fe) availability critically limits diatom distribution and productivity in vast regions of the modern ocean, such as open-ocean, high nutrient low chlorophyll areas and coastal regimes characterized as Fe limitation 'mosaics'. While unique strategies of Fe uptake and storage confer competitive advantages to pennate diatoms, the molecular determinants of low Fe acclimation are largely unknown in centric diatoms. We combined genome-wide and targeted comparative transcriptomic analysis with diagnostic biochemistry and in vivo cell staining as a platform to identify the suite of genes involved in acclimation to Fe and associated oxidative stress in Thalassiosira pseudonana. A total of 1312 genes, nearly 12% of the total genome content, responded to Fe starvation in growing cells characterized by low photosynthetic efficiency and enhanced oxidative stress, caspase activity and metacaspase expression. While 82% of the most highly upregulated genes were also represented in EST libraries derived from diverse diatoms grown under various stress conditions (e.g. silicon, CO(2) and nitrogen limitation), our analysis suggests that T. pseudonana mounts a unique molecular response to Fe starvation that includes a number of genes distinct from those of the model pennate diatom, Phaeodactylum tricornutum, which diverged ~90 million years ago. Homologues to ~50% of the upregulated genes were also identified in a metatranscriptome of eukaryotic phytoplankton communities from a chronically Fe-limited region in the Northeast Pacific. Furthermore, we provide experimental evidence that a subset of putative death-related genes participate in the cellular acclimation to low Fe and associated oxidative damage, suggesting that they co-evolved with other metabolic pathways and play adaptive roles in the success of diatoms.

Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis.

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²(+) influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²(+) import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca²(+) increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²(+) entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr549 was critical for the prolonged Ca²(+) entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.