Fluorescence microscopic evidence for intracellular silicon storage pools in diatoms and their partial separation

Diatoms are unicellular algae that occur ubiquitously in almost every salt- and freshwater habitat on earth. Silicic acid is taken up from the environment and stored in intracellular silicon storage pools (SSPs) to form species specific silica based cell walls (frustules). During biosilica formation in the silicon deposition vesicle (SDV), the silicon is transported from SSPs into the SDV. The present contribution describes the fluorescence microscopic visualization and quantification of silica solely formed from SSPs under silicon starvation. Labeling with the fluorescence marker PDMPO demonstrates continuing cell growth and cell division without silicon uptake from the environment. The SSPs of Cyclotella nana may account for up to 1/3 of total cellular silicon and intracellular concentrations up to ca. 540 mM, significantly beyond the saturation level (2 mM). The chemical state and localization of SSPs within cells are yet unclear. Here, we report a method for subcellular fractionation of C. nana by density gradient centrifugation. Quantification of cytoplasmic silicon yields 3 to 10% of the total cellular silicon corresponding to intracellular concentrations of ca. 50 to 160 mM. Labeling with NMR active 29Si shows that this silicon occurs as condensed silica, i.e., silicic acid is converted into condensed silica after uptake. However, the detected intracellular silicon concentrations are lower than the value of ca. 540 mM predicted by the fluorescence experiments. It is thus concluded that the missing SSP silicon is either present in the form of large aggregates or compartments. Alternatively, they may also be bound to cell wall biosilica.