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

Multiparametric Analyses Reveal the pH-Dependence of Silicon Biomineralization in Diatoms

International audienceDiatoms, 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 metabolis

Plataforma logística: estudo da viabilidade de implantação em Juiz de Fora (MG) via caracterização das mercadorias transportadas entre Minas Gerais e Rio de Janeiro

The logistic development induces the construction of distributioncenters that facilitate the product flow. This process leads to the building of whatis known as logistic platforms. This paper analyzes the possibility of developinga logistic platform in Juiz de Fora (MG) based on the main characteristics ofgoods transported between Minas Gerais state and Rio de Janeiro state byhighways and railways that pass along Juiz de Fora municipality. After verifyingthat there is a significant international and inter-regional trade flow and based onthe characteristics of the transported goods, it is possible to suggest that there isrole for a construction of a logistic platform at Juiz de Fora. This platform shalldeal with solid goods with high weight and low aggregated value.Com o desenvolvimento da logística, torna-se importante a construçãode centros de distribuição facilitadores do fluxo de produtos. Assim,surgem as Plataformas Logísticas. Neste contexto, o artigo busca analisar aviabilidade de implantação de uma Plataforma Logística em Juiz de Fora(MG) à luz da caracterização das mercadorias transportadas entre os Estadosde Minas Gerais e Rio de Janeiro que utilizam as vias de transporte rodoviárioe ferroviário que perpassam a cidade. Após a verificação da existência de umfluxo comercial internacional e inter-regional significativo e a caracterizaçãodos produtos transportados, sugere-se a implantação, em Juiz de Fora, deuma Plataforma Logística de produtos sólidos de grande peso e baixo valoragregado

Influence of external pH on intracellular pH.

<p><i>Thalassiosira weissflogii</i> cells were loaded with BCECF-AM at different external pH values (pHe). Ratiometric emission (with excitation at 485 and 436 nm) was used to calculate intracellular pH values (pHi). The relation between pHe and pHi measurements was fitted to a linear regression (<i>r²</i> = 0.306; Fisher's, <i>p</i><0.0001), and corresponds to 20≤<i>n</i>≤30 measurements.</p

Influence of the environmental pH on valve morphometry.

<p>(<b>A</b>) TEM image of a valve showing the different traits measured: valve width (<i>W</i>), number (<i>N</i>) and distance (<i>cp</i>) between the fultoportulae present in the central region, and distance between rimoportulae (<i>rp</i>). Scale bar: 5 µm. The right panel corresponds to a higher magnification (64,000×) showing an array of pores and the computed Voronoi diagram (<i>dark blue lines</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046722#pone.0046722.s012" target="_blank">Methods S1</a>). We measured pore radius (<i>R</i>), the average distance between two adjacent pores (<i>d1</i>), the width of groups of pores, also known as semi-continuous cribrum (<i>D</i>), and the width of radial ribs (<i>d2</i>). (<b>B</b>) Boxplot showing variations in pore radiuses (<i>R</i>) (971≤<i>n</i>≤3551) as a function of external pH (pHe). (<b>C</b>) Boxplot showing the influence of pHe on overall valve porosity (<i>r</i>) (7≤<i>n</i>≤21).</p

Influence of pHe on the kinetics of valve formation.

<p>(<b>A</b>) The initial phase of valve formation is composed of two phases: an exponential evolution of the HCK-123 signal (<i>t</i>_Exp) that ends at a maximum named F<i>₁</i>, followed by a decay phase that ends at the minimum (<i>t</i>_Dec), named F<i>₂</i>. During the first phase we fitted the data to an exponential and determined the slope (<i>k</i>) and the coordinates on the y-axis (F<i>₀</i>). (<b>B</b>) Influence of extracellular pH on <i>k</i>. In the 6.4–8.2 pH range, data were fitted to an exponential (y = 1.1e-3.e^0.32×; <i>r²</i> = 0.98). (<b>C</b>) Variation of HCK-123 concentrations as a function of external pH during the exponential (F<i>₁</i>-F<i>₀</i>) phase. In the 6.4–8.2 pH range, data were fitted to an exponential (y = 1e-4.e^1.50×; <i>r²</i> = 0.98). (<b>D</b>) Variation of HCK-123 concentrations during the decay (F<i>₁</i>-F<i>₂</i>) phase. In the 6.4–8.2 pH range, data were fitted to an exponential (y = 5e-5.e^1.47×; <i>r²</i> = 0.95). Data are from 7 to 92 single-cell measurements.</p

Measurement of valve formation dynamics at the single-cell level.

<p>(<b>A</b>) The valve formation process can be separated into several phases: an initiation phase, followed by valve formation and morphogenesis, then by valve exocytosis and finally by daughter-cell separation. The two images correspond to the DIC (digital image correlation) and Z-projections of HCK-123 fluorescence (<i>green</i>). In green we can visualize newly-synthesized valves labeled with the silica-associated dye (HCK-123). Scale bar (<i>black</i>): 10 µm. (<b>B</b>) Schematic representation of the microfluidic device used to record valve formation. During valve formation, light intensity, temperature and renewal of the medium were controlled. (<b>C</b>) To quantify HCK-123 fluorescence in individual cells, we developed new software for cell-tracking and local background estimation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046722#pone.0046722.s012" target="_blank">Methods S1</a>). Images at 4 different times are presented. Apparent cell motility is caused by the liquid flow. (<b>D</b>) To precisely quantify fluorescence in each cell we developed a new shape extraction method. The original image was denoised with the TV-means algorithm, leading to a much cleaner image. Then, among the level lines (iso-intensity curves) of the image enclosing the known center of the cell, we considered that the cell boundary was the line with the sharpest contrast (<i>L</i>). This enabled us to compute cell area <i>A(L)</i> as the area of the region enclosed by <i>L</i>), and its width <i>W(L)</i>, defined as the minimum width of a band containing <i>L</i>.</p