Suppression subtraction hybridization analysis revealed regulation of some cell cycle and toxin genes in Alexandrium catenella by phosphate limitation

Molecular mechanisms regulating responses to phosphate (P) limitation in dinoflagellates are poorly understood. Here we investigated gene differential expression Alexandrium catenella strain ATHK using suppression subtractive hybridization (SSH) and reverse-transcription quantitative PCR (qRT-PCR) techniques. SSH revealed a score of genes responded to P-limitation, and comparison with EST sequencing results indicated superior efficiency of SSH in identifying differentially expressed genes. Among the P-limitation responsive genes were the up-regulated stress-related genes, the down-regulated phosphorus-transferring genes, and a number of other genes previously undocumented for dinoflagellates. Most notably, the previously undocumented cell cycle inhibitory regulator fizzy/cell division cycle 20-related protein gene was up-regulated while putatively cell cycle promoting genes (e.g. calcium-dependent protein kinase) down-regulated, consistent with the decrease in growth rate and cell cycle arrest in G1 phase under P-limitation observed microscopically and flow cytometrically respectively. Besides, both SSH and qRT-PCR showed that saxitoxin related genes, menaquinone biosynthesis methyltransferase, an unknown gene and some other genes were also significantly induced by P-limitation. Our results demonstrate the utility of SSH in gene regulation research and provide a set of promising genes for further studies to understand the regulation of cell division (and hence population growth) and toxin production by P-limitation in A. catenella and likely other dinoflagellates.