Evidence for a Regulatory Role of Diatom Silicon Transporters in Cellular Silicon Responses

Abstract

The utilization of silicon by diatoms has both global and small-scale implications, from oceanic primary productivity to nanotechnological applications of their silica cell walls. The sensing and transport of silicic acid are key aspects of understanding diatom silicon utilization. At low silicic acid concentrations (<30 M), transport mainly occurs through silicic acid transport proteins (SITs), and at higher concentrations it occurs through diffusion. Previous analyses of the SITs were done either in heterologous systems or without a distinction between individual SITs. In the present study, we examined individual SITs in Thalassiosira pseudonana in terms of transcript and protein abundance in response to different silicic acid regimes and examined knockdown lines to evaluate the role of the SITs in transport, silica incorporation, and lipid accumulation resulting from silicon starvation. SIT1 and SIT2 were localized in the plasma membrane, and protein levels were generally inversely correlated with cellular silicon needs, with a distinct response being found when the two SITs were compared. We developed highly effective approaches for RNA interference and antisense knockdowns, the first such approaches developed for a centric diatom. SIT knockdown differentially affected the uptake of silicon and the incorporation of silicic acid and resulted in the induction of lipid accumulation under silicon starvation conditions far earlier than in the wild-type cells, suggesting that the cells were artificially sensing silicon limitation. The data suggest that the transport role of the SITs is relatively minor under conditions with sufficient silicic acid. Their primary role is to sense silicic acid levels to evaluate whether the cell can proceed with its cell wall formation and division processes. Silicon, a member of group 14 of the periodic table containing carbon, germanium, and lead, is one of the most abundant elements (27.7%) of the Earth's crust second only to oxygen (46.6%) (1). Dissolved silica in the form of orthosilicic acid, Si(OH) 4 , derived from the weathering of the Earth's crust (2), is an essential nutrient for a few groups of organisms of marine and freshwater environments, such as diatoms, radiolaria, silicoflagellates, and sponges (3). Diatoms require a large quantity of silicon, which is biomineralized to form their unique siliceous cell wall (frustule) in various shapes and morphologies with highly intricate and ornate architectures. Diatoms contribute about 40% of the total ocean organic carbon production, and they dominate ocean Si production (4, 5). Though silicon does not appear to be directly involved in cellular metabolic processes, diatoms require silicon for progression of cell cycle events, such as cell division and DNA replication (5-8). Silicon requirements are tightly coupled to cell cycle progression, a unique feature of diatoms (7). Deprivation of Si arrests the cell cycle at G 1 , G 2 , or M phase, depending upon the diatom species. In Thalassiosira pseudonana, upon Si depletion the ...

Transcript level coordination of carbon pathways during silicon starvation‐induced lipid accumulation in the diatom Thalassiosira pseudonana

“…Recent work by Shrestha et al . (2015) show that knockdown of silicic acid transporters (SITs) results in an earlier onset in lipid accumulation under silicon starvation than wild‐type T. pseudonana, indicating the presence of a silicon‐sensing mechanism. We identified genes that are highly up‐regulated and coexpressed with SIT1 during silicon starvation, including a protein kinase (Thaps3_14322) and a hypothetical protein (Thaps3_9619).…”

Competition between Silicifiers and Non-silicifiers in the Past and Present Ocean and Its Evolutionary Impacts

“…Transcriptional analysis of these SIT types under varying silicic acid concentrations demonstrated that the SITα genes were highly expressed, and their expression levels responded to silicic acid availability, while the SITβ genes were always expressed at a low level, irrespective of the environmental silicic acid concentration. This closely resembles the situation in diatoms (see above), where some SIT genes are highly expressed and silicon-responsive, while other types have low expression unresponsive to silicon, suggesting that SITα and SITβ have evolved different roles, possibly as specialized transporters or silicon sensors (Thamatrakoln and Hildebrand, 2007;Shrestha and Hildebrand, 2015). The similar diversification and subfunctionalization of SITs in parallel in both choanoflagellates and diatoms is proposed to be an example of convergent evolution in response to increased competition for silicic acid.…”

“…At lower silicic acid levels (≤30 µM), the majority of uptake is by SIT-mediated active transport, while at higher concentrations silicic acid enters the cell by diffusion (Thamatrakoln and Hildebrand, 2008;Shrestha and Hildebrand, 2015). The concentration gradient is created by binding of silicic acid to intracellular binding components in the cytoplasm, the character of which remains unknown (Thamatrakoln and Hildebrand, 2008;Spinde et al, 2011).…”

“…Diatom species generally possess multiple SIT genes (Hildebrand et al, 1998). These paralogs display differences in expression levels, the timing of their expression in the cell cycle, protein abundances and their sub-cellular localization (Thamatrakoln and Hildebrand, 2007;Sapriel et al, 2009;Shrestha et al, 2012;Shrestha and Hildebrand, 2015). These differences are believed to allow neofunctionalization of each SIT, evolving roles as silicon sensors, silicon transporters, targeting of different SIT proteins to specific cellular locations (Shrestha and Hildebrand, 2015), specialization for the uptake of certain silicon species (Sapriel et al, 2009) or for differential characteristics of substrate affinity versus transport capacity.…”

Editing of the urease gene by CRISPR-Cas in the diatomThalassiosira pseudonana

“…The genus has multiple biotechnology applications [8,21,22], and although gene silencing has been established, a method to easily and efficiently knock-out and edit genes and the entire genome would be highly advantageous. The genus Thalassiosira is among the top 10 genera of diatoms in the World's Ocean in terms of ribotype (V9 of 18S) diversity and abundance [23] and the species T. pseudonana is a model for understanding the mechanisms behind silicification [24][25][26].…”