Hallmarks of the dynamic responses to silica in Thalassiosira pseudonana - Identification, characterization and classification of signature genes and their corresponding motifs

The diatom cell wall, or frustule, is a highly complex, three-dimensional structure consisting of nanopatterned silica as well as proteins and other organic components. While some key components have been identified, knowledge on frustule biosynthesis is still fragmented. The model diatom Thalassiosira pseudonana was subjected to silicon (Si) shift-up and shift-down situations. Cellular and molecular signatures, dynamic changes and co-regulated clusters representing the hallmarks of cellular and molecular responses to changing Si availabilities were characterised. 62 silaffin-like proteins (SFLPs), two kinases and a novel family of putatively frustule-associated transmembrane proteins induced by Si shift-up and a possible role in frustule biosynthesis were identified. A separate cluster analysis performed on all significantly regulated SFLPs, as well as fourteen new proteins with silaffin-like motifs, resulted in the classification of silaffins, cingulins and SFLPs into distinct clusters. The genes in the Si-responsive clusters are in general highly divergent, but positive selection does not seem to be the driver behind this variability. This study provides a high-resolution map over transcriptional responses to changes in Si availability in T. pseudonana. Hallmark Si-responsive genes are identified, characteristic motifs and domains are classified, and taxonomic and evolutionary implications outlined and discussed. Thalassiosira pseudonana (CCAP 1085/2) was grown in axenic cultures at 20°C, illuminated with 130 µmol photons m-2 s-1 in light:dark cycles of 16:8 hours. Both experiments (shift-up and shift-down) were initiated at least 4 hours after beginning of the light phase, and all consecutive samplings were performed within the same light period. The Si-depleted medium contained 0.7 µM Si, and was used to prepare the experimental medium for the shift-down experiment following the modified f/2 recipe, but with no Si added. For the shift-up experiment, 1000 mL pre-cultures were cultivated as batch cultures in medium with f/4-levels of Si (approximately 55-60 uM Si, including a seawater background of 4 uM) for three weeks prior to the experiment; the cultures were diluted with fresh medium when the Si was depleted. The silicon shift-down experiment was sampled before centrifugation (-2 h), and thereafter, samples were taken every hour or half hour for 7.5 hours after resuspension in silicate-free medium. Samples from time point 72 h were also included. The time points -2, 0.33, 1, 2, 4, 7.5, and 72 hours were selected for further analyses. For the silicon shift-up experiment, cultures were sampled right before Si addition, and then every hour or every half hour as the biomass began to increase. Based on the time course of the culture, the time points -0.25 (-15 min, control), 0.25, 0.5, 1, 2, 4, 6, and 8 hours relative to the silicate shift-up were selected for further analyses.