Multiparametric analyses reveal the pH-dependence of silicon biomineralization in diatoms.

Diatoms, the major contributors of the global biogenic silica cycle in modern oceans, account for about 40% of global marine primary productivity. They are an important component of the biological pump in the ocean, and their assemblage can be used as useful climate proxies; it is therefore critical to better understand the changes induced by environmental pH on their physiology, silicification capability and morphology. Here, we show that external pH influences cell growth of the ubiquitous diatom Thalassiosira weissflogii, and modifies intracellular silicic acid and biogenic silica contents per cell. Measurements at the single-cell level reveal that extracellular pH modifications lead to intracellular acidosis. To further understand how variations of the acid-base balance affect silicon metabolism and theca formation, we developed novel imaging techniques to measure the dynamics of valve formation. We demonstrate that the kinetics of valve morphogenesis, at least in the early stages, depends on pH. Analytical modeling results suggest that acidic conditions alter the dynamics of the expansion of the vesicles within which silica polymerization occurs, and probably its internal pH. Morphological analysis of valve patterns reveals that acidification also reduces the dimension of the nanometric pores present on the valves, and concurrently overall valve porosity. Variations in the valve silica network seem to be more correlated to the dynamics and the regulation of the morphogenesis process than the silicon incorporation rate. These multiparametric analyses from single-cell to cell-population levels demonstrate that several higher-level processes are sensitive to the acid-base balance in diatoms, and its regulation is a key factor for the control of pattern formation and silicon metabolism.

Genome-wide transcriptome analyses of silicon metabolism in Phaeodactylum tricornutum reveal the multilevel regulation of silicic acid transporters.

BACKGROUND: Diatoms are largely responsible for production of biogenic silica in the global ocean. However, in surface seawater, Si(OH)(4) can be a major limiting factor for diatom productivity. Analyzing at the global scale the genes networks involved in Si transport and metabolism is critical in order to elucidate Si biomineralization, and to understand diatoms contribution to biogeochemical cycles. METHODOLOGY/PRINCIPAL FINDINGS: Using whole genome expression analyses we evaluated the transcriptional response to Si availability for the model species Phaeodactylum tricornutum. Among the differentially regulated genes we found genes involved in glutamine-nitrogen pathways, encoding putative extracellular matrix components, or involved in iron regulation. Some of these compounds may be good candidates for intracellular intermediates involved in silicic acid storage and/or intracellular transport, which are very important processes that remain mysterious in diatoms. Expression analyses and localization studies gave the first picture of the spatial distribution of a silicic acid transporter in a diatom model species, and support the existence of transcriptional and post-transcriptional regulations. CONCLUSIONS/SIGNIFICANCE: Our global analyses revealed that about one fourth of the differentially expressed genes are organized in clusters, underlying a possible evolution of P. tricornutum genome, and perhaps other pennate diatoms, toward a better optimization of its response to variable environmental stimuli. High fitness and adaptation of diatoms to various Si levels in marine environments might arise in part by global regulations from gene (expression level) to genomic (organization in clusters, dosage compensation by gene duplication), and by post-transcriptional regulation and spatial distribution of SIT proteins.

Plasticity and robustness of pattern formation in the model diatom Phaeodactylum tricornutum.

Understanding the morphogenesis of mineralized structures found in shells, bones, teeth, spicules and plant cell walls is difficult because of the complexities underlying biomineralization, and the requirement of accurate models for pattern formation. Here, we investigated the spatial and temporal development of siliceous structures found in a model diatom species, Phaeodactylum tricornutum, for which the entire genome has been sequenced and transformation is routine. Analyses of pattern formation revealed that the process of silicification starts from a 'pi-like' structure that controls the spatial organization of a sternum upon which regular instabilities are initiated and developed. Detailed analyses also demonstrate that morphogenesis of silica is nonuniform. We also tested the sensitivity of pattern formation to perturbation of proton pumps, and found that selective inhibitors of H(+)-V-ATPases affect silica biomineralization both quantitatively and qualitatively. Morphometric analyses of valves purified from isogenic populations of cells show that the morphometric noise of several traits is under exquisite regulation, explaining why the overall valve pattern is reproducibly maintained. Altogether our analyses demonstrate that silica morphogenesis is a robust but nonuniform process, and allow us to propose a model for the dynamic growth of materials within a spatially controlled geometry.

New tools for labeling silica in living diatoms.

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.