Silicon uptake in diatoms revisited: a model for saturable and nonsaturable uptake kinetics and the role of silicon transporters

Abstract

The silicic acid uptake kinetics of diatoms were studied to provide a mechanistic explanation for previous work demonstrating both nonsaturable and Michaelis-Menten-type saturable uptake. Using (68)Ge(OH)(4) as a radiotracer for Si(OH)(4), we showed a time-dependent transition from nonsaturable to saturable uptake kinetics in multiple diatom species. In cells grown under silicon (Si)-replete conditions, Si(OH)(4) uptake was initially nonsaturable but became saturable over time. Cells prestarved for Si for 24 h exhibited immediate saturable kinetics. Data suggest nonsaturability was due to surge uptake when intracellular Si pool capacity was high, and saturability occurred when equilibrium was achieved between pool capacity and cell wall silica incorporation. In Thalassiosira pseudonana at low Si(OH)(4) concentrations, uptake followed sigmoidal kinetics, indicating regulation by an allosteric mechanism. Competition of Si(OH)(4) uptake with Ge(OH)(4) suggested uptake at low Si(OH)(4) concentrations was mediated by Si transporters. At high Si(OH)(4), competition experiments and nonsaturability indicated uptake was not carrier mediated and occurred by diffusion. Zinc did not appear to be directly involved in Si(OH)(4) uptake, in contrast to a previous suggestion. A model for Si(OH)(4) uptake in diatoms is presented that proposes two control mechanisms: active transport by Si transporters at low Si(OH)(4) and diffusional transport controlled by the capacity of intracellular pools in relation to cell wall silica incorporation at high Si(OH)(4). The model integrates kinetic and equilibrium components of diatom Si(OH)(4) uptake and consistently explains results in this and previous investigations.

Figure 1.

Effect of incubation time on Si(OH)₄ uptake kinetics in T. pseudonana. Uptake rates were measured in cells incubated in various silicate concentrations for 2 min, 10 min, 30 min, 1 h, 2 h, or 3 h. Curves represent fitted Michaelis-Menten hyperbolas obtained by nonlinear regression. For 8 to 30 μmol L⁻¹ Si(OH)₄ concentrations, the average of duplicates is plotted. Error bars are se. Inset graph in 2-min uptake represents Si(OH)₄ uptake at concentrations up to 500 μmol L⁻¹. To compare rates between experiments and with past data, all uptake rates were expressed in hours even when short-term (e.g. minutes) uptake was measured.

Figure 2.

Detailed analysis of Si(OH)₄ uptake. Data were fit by nonlinear regression using a sigmoidal dose-response model. The mean with se is shown. A, Short-term (2 min) uptake at 1 to 30 μmol L⁻¹ Si(OH)₄. Inset shows uptake up to 100 μmol L⁻¹ (n = 11). B, Short-term uptake measured every 2 μmol L⁻¹ Si(OH)₄ up to 30 μmol L⁻¹ (n = 7). C, Long-term uptake (2 and 3 h) below 30 μmol L⁻¹.

Figure 3.

A, Effect of zinc chelators on Si(OH)₄ uptake. Membrane-permeable chelator TPEN and membrane-impermeable chelator Na₂EDTA were added to cells at 50 μmol L⁻¹. Si(OH)₄ uptake in 15 μmol L⁻¹ Si(OH)₄ was monitored after 30 min. Control cells had no addition, and another control had an equal volume of DMSO as used for the TPEN solution. The mean of triplicate measurements is shown along with se. Cell density was monitored in cultures grown in ASW (control) or ASW + TPEN after the addition of Ca, Fe, or Zn (inset). The mean of duplicates is shown with sd. B, Comparison of short-term (2 min) Si(OH)₄ uptake using the standard protocol (white circles, solid line) and with a constant ratio of unlabeled Si(OH)₄ to Ge(OH)₄ (black squares, dashed line). Inset shows the slope of both conditions between 1 and 30 μmol L⁻¹; slope above 30 μmol L⁻¹ is evident in the original plot.

Figure 4.

Si(OH)₄ uptake kinetics of N. pelliculosa FW. All data were fit by nonlinear regression using Michaelis-Menten hyperbolas. A, Left graph shows short-term (2 min) uptake kinetics using the same method used for T. pseudonana in Figures 1 and 2. Right graph is uptake kinetics for the same cells measured after 30 min. B, In contrast, cells here were maintained in Si-free medium for 24 h prior to measuring short-term uptake kinetics (2 min). Error bars for 8 to 30 μmol L⁻¹ Si(OH)₄ concentrations are se of two replicates. Values for K_s (in micromoles per liter) and V_max (in femtomoles per cell per hour) were calculated using GraphPad Prism 4.

Figure 5.

Measurement of intracellular Si(OH)₄ pools under different growth conditions. A, Pools were measured in exponentially growing (Exp) cells and then over time in cells spiked with 100 μmol L⁻¹ Si(OH)₄ after a brief Si starvation (5–10 min). The mean of duplicate measurements is shown with se. B, Pools measured in exponentially growing cultures (n = 13) compared to Si(OH)₄ levels in the medium. The mean of duplicate measurements is shown with se.

Figure 6.

Maximum Si(OH)₄ uptake rates and time to achieve saturable kinetics for different diatom species. A, Maximum rate of Si(OH)₄ uptake for different diatom species after 2 min, 10 min, 30 min, 1 h, 2 h, or 3 h. T. pseudonana, C. fusiformis, C. gracilis, P. tricornutum, and N. pelliculosa FW were incubated with 100 μmol L⁻¹ Si(OH)₄. T. weissflogii, N. pelliculosa M, and N. alba were incubated with 30 μmol L⁻¹ Si(OH)₄. Break in y axis is shown. B, Rate of decrease in uptake for different species based on percent of maximal uptake at 2 min over time. Exponential decay curves were fit; symbols denote the different species.

Figure 7.

Ratio of cell wall silica to intracellular soluble Si pools for exponentially growing diatom species used in this study. See “Materials and Methods” for experimental details.

Figure 8.

Proposed model of Si uptake in diatoms. In each image, a diatom cell is represented as a rectangular box and cell wall silica (i.e. the silica deposition vesicle, SDV) is represented as a gray elongated oval. Black dots represent Si(OH)₄. Horseshoe-shaped structures represent intracellular Si-binding components that data suggest are present but have yet to be identified. Arrows denote direction of transport with magnitude indicated by their thickness. Black arrows denote diffusion-mediated uptake, and broken arrows represent SIT-mediated transport. Hatched arrows show movement of Si(OH)₄ into the SDV. Stylized graphs of uptake kinetics are shown for C and E. A, In exponentially growing cells, equilibration is achieved between uptake rate, intracellular pools, and cell wall silica incorporation. Uptake is internally controlled. B, After a brief (5–10 min) incubation in Si-free medium, levels of intracellular-binding component have delivered Si(OH)₄ to the SDV and are predominantly in the uncomplexed state. C, Upon Si(OH)₄ replenishment, cells are able to accommodate surge uptake mediated by diffusion at high Si(OH)₄, and nonsaturable uptake kinetics are observed. Biphasic curves are seen because, at low Si(OH)₄ concentrations, SITs are still capable of mediating uptake. In T. pseudonana, this results in sigmoidal kinetics. SIT-mediated efflux aids in equilibration. D, After time (approximately 1 h in T. pseudonana), equilibration is re-established between the level of binding component and the rate of silica incorporation, and uptake becomes internally controlled. E, During long-term (24 h) Si starvation, the level of binding component becomes reduced. F, Upon Si(OH)₄ replenishment, intracellular capacity is low, and cells are not able to accommodate surge uptake; thus, Michaelis-Menten type saturation is observed.