Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana

Abstract

Background: Silicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays.

Results: Three known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes. In terms of diatom cell wall formation, thus far only cell surface proteins and proteins tightly associated with silica have been characterized. Our analysis has identified new genes potentially involved in silica formation, and other genes potentially involved in signaling, trafficking, protein degradation, glycosylation and transport, which provides a larger-scale picture of the processes involved. During silicon starvation, an overrepresentation of transcription and translation related genes were up-regulated, indicating that T. pseudonana is poised to rapidly recover from silicon starvation and resume cell cycle progression upon silicon replenishment. This is in contrast to other types of limitation, and provides the first molecular data explaining the well-established environmental response of diatoms to grow as blooms and to out-compete other classes of microalgae for growth. Comparison of our data with a previous diatom cell cycle analysis indicates that assignment of the cell cycle specific stage of particular cyclins and cyclin dependent kinases should be re-evaluated. Finally, genes co-varying in expression with the SITs enabled identification of a new class of diatom-specific proteins containing a unique domain, and a putative silicon efflux protein.

Conclusions: Analysis of the T. pseudonana microarray data has provided a wealth of new genes to investigate previously uncharacterized cellular phenomenon related to silicon metabolism, silicon's interaction with cellular components, and environmental responses to silicon.

Figure 1

Diatom cell wall structures and components. Left, SEMs showing the diversity of diatom valve structures from different species. Scale bars a, k) 1 μm, b) 5 μm c, f) 10 μm d) 500 nm e, g, h, j, l) 2 μm, i) 50 μm. Center, diagram of a diatom cell during valve formation. Right, diagram of a dividing diatom cell highlighting major aspects of silicon’s interaction with the cell or valve formation (colored boxes) that are major focus areas of this study. Specific components are described in detail in the text.

Figure 2

Bar diagram of functional category enrichment analysis (Fisher's Exact Test) of SLRGs (Test Set) and total data set (Reference Set). Y-axis represents significant enrichment of GO terms and X-axis shows the relative frequency of the term.

Figure 3

Expression pattern of girdle-band associated cingulins compared with Tpsil1. Two cingulins W1 and W2 were present in SLRG dataset.

Figure 4

Hierarchical clustered expression profile of 485 silaffin-like response genes (SLRGs), and specific responses of the Tpsil3 cluster. Left) Heatmap of the SLRG data. Columns correspond to log2ratio (fold change) of the time points (2, 4, 7, 8 and 9 h) relative to 0 h of a synchronized cell culture. The intensities of the colors indicate the magnitude of up-regulation (red) and down regulation (green). Black indicates no change. The Tpsil3 cluster is indicated by the blue bar. Right) upper is the expression profile of Tpsil3 and lower are the expression profiles of genes in the Tpsil3 cluster showing expression level fold changes.

Figure 5

Partial amino acid sequences of two genes of the silaffin cluster.A) Thaps3_24597 has three complete 53 aa repeats and one partial repeat. The sequence was verified by sequencing cDNA and one extra complete repeat than predicted in the Thaps vers. 3 genome database was found. B) Thaps3_ 25828 C-terminal sequence has three repeats and KXXK motifs. KXXK motifs are denoted by asterisks. MSMSM motifs are overlined.

Figure 6

Bar diagram of functional category enrichment analysis of SSRGs (Test Set) and total data set (Reference Set). Y-axis represents significant enrichment of GO terms and X-axis shows the relative frequency of the term.

Figure 7

Expression pattern of SIT 1, 2 and 3.

Figure 8

Multiple alignment of Thaps3_21292 and plant silicon efflux transporters. Sequence annotations: Rice,BAF73750.1; Maize, NP_001183945.1; Barley, BAH84976.1; T. pseudonana, Thaps3_21292; P. tricornutum, Phatr2_33218.