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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Silicon Uptake in Diatoms Revisited: A Model for Saturable and Nonsaturable Uptake Kinetics and the Role of Silicon Transporters^1,[OA]

Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, California 92093–0202

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 ⁶⁸Ge(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.

Diatoms are one of the largest groups of silicifying organisms, and most species have an obligate requirement for silicon (Si) for cell wall formation. Si transporters, or SITs, are specific membrane-associated proteins shown to transport Si(OH)₄ across lipid bilayer membranes (Hildebrand et al., 1997, 1998). SITs have been identified in numerous diatom species (Grachev et al., 2002; Sherbakova et al., 2005; Thamatrakoln et al., 2006) as well as in the Chrysophytes Synura petersenii and Ochromonas ovalis (Likoshway et al., 2006). SITs have also been identified in plants, but these proteins are homologous to plant aquaporins or bacterial arsenic effluxers and share no similarity with diatom SITs (Ma et al., 2004; Ma et al., 2007). Mechanistic models for Si transport in diatoms have recently been proposed (Sherbakova et al., 2005; Thamatrakoln et al., 2006), including one (Sherbakova et al., 2005) with a hypothesis for the proposed involvement of zinc in Si(OH)₄ uptake (Rueter and Morel, 1981).

In diatoms, Si(OH)₄ uptake has been typically characterized by Michaelis-Menten-type saturation kinetics, although nonsaturable and biphasic kinetics have occasionally been observed (Table I ). Many kinetics studies on diatom Si(OH)₄ uptake utilized long-term incubations; however, it is well established that intrinsic kinetic properties need to be measured in the short term prior to the influence of equilibrium effects. Equilibrium factors influencing Si(OH)₄ uptake in diatoms include low extracellular Si(OH)₄ concentrations (<100 µM; Tréguer et al., 1995; Martin-Jézéquel et al., 2000); high intracellular concentrations (up to hundreds of millimolars); and different forms of Si, extracellular being silicic acid or silicate, intracellular thought to consist of silicic acid complexed with organics (Azam et al., 1974; Sullivan, 1979), and the solid form of silica in the cell wall (Martin-Jézéquel et al., 2000). Unlike other nutrients, silicic acid has a unique chemistry in that it autopolymerizes into silica at concentrations >2 mM at neutral pH (Iler, 1979), but intracellular pools in diatoms have been measured well above silica saturation (19–340 mM; Martin-Jézéquel et al., 2000). Electron spectroscopic imaging indicates intracellular Si is not sequestered in vesicles (Rogerson et al., 1987), and other data suggest that maintenance of supersaturated levels of silicic acid is likely through an association of intracellular Si with as-yet-uncharacterized organic components (Azam et al., 1974; Sullivan, 1979; Hildebrand, 2000; Hildebrand and Wetherbee, 2003). The balance between these binding components and free Si(OH)₄ will also influence the equilibrium state of Si(OH)₄. Si efflux is an often overlooked and underappreciated aspect of balancing the overall cellular Si budget, with net uptake involving both uptake and efflux (Azam et al., 1974; Sullivan, 1976; Milligan et al., 2004).

The described model for Si transport deals mainly with equilibrium processes and does not completely explain observed kinetic parameters (Table I). In each study (Table I) documenting Michaelis-Menten-type saturation kinetics for Si(OH)₄ uptake in diatoms, either (1) cultures were maintained in Si-free medium for an extensive period of time (24 h) prior to measuring uptake; (2) uptake was measured over long (hours) incubation times; or (3) low Si(OH)₄ concentrations were used (Table I). When these conditions were not followed, nonsaturable uptake kinetics were observed (Table I), but explanations for nonsaturable kinetics have not been provided.

To correlate kinetic observations with equilibrium processes in the model for Si(OH)₄ transport, we measured both short-term (minutes) and long-term (hours) uptake in different diatom species using the radiotracer analog of silicic acid, ⁶⁸Ge(OH)₄. In addition, we determined and could control conditions to produce saturable or nonsaturable Si(OH)₄ uptake kinetics. Based on these data and previous studies, a revised model of Si(OH)₄ uptake in diatoms, in which both carrier-mediated and diffusional transport occurs, was developed to provide explanations for observed kinetics curves based on equilibrium factors.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Si(OH)₄ Uptake in Thalassiosira pseudonana Transitioned from Nonsaturable to Saturable Kinetics over Time

In previous work, incubation time was one variable distinguishing Michaelis-Menten saturating from nonsaturating or biphasic Si(OH)₄ uptake kinetics in diatoms (Table I). To determine the effect of incubation time on Si(OH)₄ uptake kinetics, uptake rates were measured on the same sample of exponentially growing T. pseudonana by incubation with various silicate concentrations and removing aliquots to measure uptake after 2 min, 10 min, 30 min, 1 h, 2 h, and 3 h. In Figure 1 , curves were fit by nonlinear regression using Michaelis-Menten hyperbolas. Kinetic parameters are listed in Table II . For Si(OH)₄ uptake after 2 and 10 min, a Michaelis-Menten hyperbola could be fit to the data, but a clear plateau was not observed and uptake appeared nonsaturable. In addition, the 95% confidence intervals for K_s [the half-saturation constant defined as the Si(OH)₄ concentration at 0.5 V_max] and V_max were extremely wide, suggesting a poor fit of the data to a Michaelis-Menten model (Table II). In a separate experiment, short-term uptake continued to increase at concentrations up to 500 µmol L^–1, and data could not be fit to a Michaelis-Menten hyperbola (data did not converge) consistent with uptake being nonsaturable (Fig. 1, 2 -min inset). Comparing Michaelis-Menten plots from 30 min to 3 h, the curves gradually sloped over, and saturation was observed between 1 and 2 h (Fig. 1). Based on measurements of Si requirements for the cell wall of T. pseudonana (Hildebrand et al., 2007), Si(OH)₄ would not be depleted in the long-term incubations, indicating that sloping over was not due to a transition to externally controlled uptake.

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   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.

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   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.

Previous experiments in T. pseudonana reported saturation at Si(OH)₄ concentrations <50 µmol L^–1 (Table I), in contrast to our results (Fig. 1, 2-min inset). To address this, uptake at Si(OH)₄ concentrations between 1 and 100 µmol L^–1 was analyzed in more detail on multiple biological and technical replicates (n = 11). Biphasic kinetics were observed, with a nonsaturable aspect above 30 µmol L^–1 (Fig. 2A, inset) and a sigmoidal dose-response curve (a Michaelis-Menten hyperbola resulted in a poor fit of the data) below 30 µmol L^–1 (Fig. 2A), with K_0.5 = 8.1 ± 1.4 µmol L^–1 and V_max = 8.4 ± 1.1 fmol cell^–1 h^–1. The Hill slope, an indicator of the degree of cooperativity (Koshland et al., 1966), was 2.7, suggesting a positively cooperative uptake system. To investigate this in more detail, short-term uptake was measured (n = 7) for Si(OH)₄ concentrations <30 µmol L^–1, at every 2 µmol L^–1. Data were fit to either a Michaelis-Menten hyperbola or a sigmoidal dose-response curve, with the latter curve (Fig. 2B) producing the better fit, with K_0.5 = 8.0 ± 0.94 µmol L^–1, V_max = 32.6 ± 2.8 fmol cell^–1 h^–1, and a Hill slope of 1.9. Long-term uptake kinetics <30 µmol L^–1 were also analyzed; in individual experiments at 2 and 3 h, uptake also showed sigmoidal kinetics (Fig. 2C), with K_0.5 = 7.2 µmol L^–1 and 5.6 µmol L^–1, respectively.

SITs Mediate Uptake at Low Si(OH)₄ Concentrations

To determine the role SITs played in uptake at different concentrations of Si(OH)₄, we tested potential inhibitors of SITs. Zinc has been proposed to be an essential component of Si transport (Rueter and Morel, 1981) by binding to a functional site on SITs and facilitating the formation of a ternary complex with silicic acid (Sherbakova et al., 2005). The effect of removing zinc on Si(OH)₄ uptake was investigated using two chelators: membrane-impermeable Na₂EDTA and zinc-specific, membrane-permeable N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Chelators used at 10-fold molar excess to zinc inhibited growth (data not shown). Growth of TPEN-inhibited cultures could be rescued by the addition of zinc but not by the addition of calcium or iron, suggesting TPEN was specifically removing zinc from the medium (Fig. 3A , inset). Chelators had no effect on Si(OH)₄ uptake rates (Fig. 3A), suggesting removal of zinc could not be used as a SIT inhibitor. Data obtained by Rueter and Morel (1981) suggesting that zinc was involved in uptake resulted from a long-term incubation experiment, suggesting secondary effects on cellular metabolism could have resulted in their observed inhibition of uptake.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   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.

Uptake Kinetics in Navicula pelliculosa FW and the Effect of Si Starvation

Another variable distinguishing Michaelis-Menten-type saturating from nonsaturating or biphasic Si(OH)₄ uptake kinetics in previous work was whether or not cells were prestarved for Si for a long time period (Table I). To test if nonsaturability of short-term Si(OH)₄ uptake (Figs. 1 and 2A) observed in T. pseudonana was due to experimental design (e.g. lack of extensive prestarvation) or whether species-specific effects were involved, Si(OH)₄ uptake was monitored in N. pelliculosa FW where short-term uptake (2 min) was previously shown to be saturable (Sullivan, 1976, 1977). To compare the effect of experimental design, Si(OH)₄ uptake was measured using both the current method (exponentially growing cells with a 5- to 10-min incubation in Si-free medium) as well as Sullivan's method (incubation of exponentially growing cells in Si-free medium for 24 h). Using the same method used in Figures 1 and 2 for T. pseudonana, Si(OH)₄ uptake was measured in N. pelliculosa FW after 2 and 30 min. For 2-min uptake, a Michaelis-Menten hyperbola resulted in a poor fit of the data (r² = 0.8557), indicating nonsaturable kinetics (Fig. 4A , left). Uptake after 30 min fit a Michaelis-Menten saturating hyperbola (r² = 0.9968) with K_s = 48.5 µmol L^–1 and V_max = 5.0 fmol cell^–1 h^–1 (Fig. 4A, right). Thus, a transition from nonsaturable to saturable kinetics occurred with increasing incubation time in exponentially grown N. pelliculosa FW, as with T. pseudonana. When cultures of N. pelliculosa FW were maintained in Si-free medium for 24 h (long-term Si starvation), short-term (2 min) uptake saturated immediately (K_s = 14.3 µmol L^–1 and V_max = 2.7 fmol cell^–1 h^–1; Fig. 4B) as previously observed (Sullivan, 1976, 1977). Short-term uptake in T. pseudonana after 24 h in Si-free medium also showed Michaelis-Menten-type saturation kinetics (data not shown), confirming this was not a species-specific effect.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   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 Ks (in micromoles per liter) and Vmax (in femtomoles per cell per hour) were calculated using GraphPad Prism 4.

Because intracellular soluble Si pool levels decrease during Si starvation (Martin-Jézéquel et al., 2000), data in Figure 4 suggested the status of intracellular pools was involved in the transition from nonsaturable to saturable kinetics. In a representative experiment in T. pseudonana (Fig. 5A ), pool levels were relatively low during exponential growth, but after 5 to 10 min in Si-free medium (as was done for the kinetics measurements), levels increased substantially within 5 min after Si(OH)₄ addition, consistent with surge uptake measured in kinetics experiments (Figs. 1 and 2A). Pools then gradually decreased (Fig. 5A). Comparison of intracellular pools (Fig. 5B) in multiple exponentially growing cultures showed relatively low levels of Si(OH)₄ (average 5.35 fmol cell^–1), even in cases where extracellular Si(OH)₄ concentrations were far higher than 100 µmol L^–1 when surge uptake occurred after processing for kinetics measurements (Fig. 5A). Thus, surge uptake was prevented under conditions of exponential growth.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   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.

A survey of different diatom species was done to determine how common nonsaturable Si(OH)₄ uptake on short time scales was and whether uptake saturated on longer time scales. Si(OH)₄ uptake was measured in different diatom species using the same method as for T. pseudonana in Figure 1. For each species tested, full kinetic curves were obtained for each incubation period, but for simplicity only V_max at the highest tested Si(OH)₄ concentration for each incubation period is shown in Figure 6A . In each species, short-term (2 min) Si(OH)₄ uptake was maximal and nonsaturable and decreased with increased time (Fig. 6A). For short-term uptake, Thalassiosira weissflogii had the highest V_max, while N. pelliculosa M had the lowest. Saturation was achieved in all species except T. weissflogii (which tended toward saturation but did not achieve it). The time required to achieve saturation varied. The percentage of uptake over time relative to the maximum at 2 min (Fig. 6B) was plotted, and exponential decay curves were used to calculate when saturation (defined as 15% of the 2-min value based on the appearance of the curve) was achieved. N. pelliculosa FW (4.8-min saturation time), Chaetoceros gracilis (10.8 min), and N. pelliculosa M (13.8 min) saturated earlier than the others, with T. pseudonana (43.3 min) taking the longest to saturate.

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   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.

Because cell wall silica incorporation can be a controlling factor over uptake through the intermediary of intracellular pools (Conway et al., 1976; Conway and Harrison, 1977; Hildebrand, 2000; Hildebrand and Wetherbee, 2003), intracellular soluble Si pool concentrations and the amount of cell wall silica were measured in each species during exponential growth to determine whether these parameters were related to the rate at which saturation was achieved. The ratio of cell wall silica to soluble intracellular pools is shown in Figure 7 . Although there was no consistent relationship between this ratio and the rate of saturation, selected examples will be discussed below.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   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.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Si(OH)₄ Uptake in T. pseudonana Occurs through Two Distinct Mechanisms

At Si(OH)₄ concentrations <30 µmol L^–1 in both short-term and long-term uptake, SITs controlled uptake, resulting in sigmoidal kinetics. Sigmoidal kinetics are diagnostic of allosteric cooperative interactions (e.g. homo- or heterodimers) and have been described for both enzymes and transporters (Hamill et al., 1999; Zelcer et al., 2003; Higgins and Linton, 2004; Suana and Ambudkar, 2007). The Hill coefficient estimates the minimal number of interacting binding sites in a positively cooperating system and suggests here that Si(OH)₄ uptake involves at least two and perhaps three binding events (Hill coefficients = 1.9–2.7). This could indicate sequential Si(OH)₄ binding or, because SITs are sodium symporters (Bhattacharyya and Volcani, 1983; Hildebrand, 2000), could indicate sequential sodium and Si(OH)₄ binding. Another type of allosteric mechanism that could explain sigmoidal kinetics is the alternating access model for transmembrane transport, where specific conformational changes promote binding at one site and affect binding at another. This mechanism has been proposed for SITs (Thamatrakoln et al., 2006) and could contribute to the allostery seen in Figure 2. Although our current data do not allow determination of the degree to which the different mechanisms described may contribute to sigmoidal kinetics, allostery may explain why the competitive inhibitor Ge(OH)₄ (Azam et al., 1974) stimulated uptake at low Si(OH)₄ concentrations (Fig. 3B). Allosteric uptake mechanisms can stimulate uptake with increasing amounts of substrate, even if substrates are competing for the same binding site.

The positive degree of cooperativity with increasing Si(OH)₄ (Fig. 2) altered SIT activity depending on extracellular Si(OH)₄ concentrations in a nonlinear way. One consequence is that SITs may limit uptake at suboptimal Si(OH)₄ concentrations, for example, if extracellular Si(OH)₄ concentrations are too low to provide sufficient precursor to complete cell wall synthesis. Darley and Volcani (1969) showed in Cylindrotheca fusiformis that a threshold level of Si(OH)₄ was required to stimulate DNA replication and cytokinesis, consistent with this hypothesis.

Uptake >30 µmol L^–1 Si(OH)₄ was nonsaturable (Fig. 1, 2-min inset) and likely mediated by simple diffusion, as opposed to facilitated diffusion (i.e. uptake through a carrier), which would be expected to saturate due to limitations in the numbers of carriers. This is supported by the lack of an effect of adding extra unlabeled Ge(OH)₄ (Fig. 3B) where the slopes of the curves >30 µmol L^–1 were nearly identical. Recently, it was proposed that Si(OH)₄ uptake in diatoms could occur by pinocytosis (Vrieling et al., 2007); however, pinocytosis is a saturable process, which is inconsistent with our data. Diffusion of Si(OH)₄, which has been previously shown to occur across a membrane (Johnson and Volcani, 1978), may seem counterintuitive in a diatom cell because intracellular concentrations are far higher than extracellular levels. However, if intracellular Si is bound by organic compounds, the chemical form is different and an inward concentration gradient for Si(OH)₄ would exist.

A Revised Model for Si(OH)₄ Uptake in Diatoms

Examination of the kinetic response to short- and long-term Si starvation (Figs. 1 and 4), and measurements of intracellular pools (Fig. 5), indicates a major controlling factor regulating the transition from nonsaturable to saturable uptake is the capacity of intracellular pools to accommodate excess soluble Si. Based on this, we propose the following model to explain the kinetic properties of diatom Si(OH)₄ uptake (Fig. 8 ). In exponentially growing cultures, intracellular Si(OH)₄ levels are relatively low, but the capacity of pools is high (Fig. 5). We propose that during the brief period (5–10 min) of Si starvation prior to the measurement of uptake, intracellular binding components continue to release their Si for incorporation, but because no extracellular Si is present, they are not recharged (Fig. 8B). Thus, when cells are subsequently incubated with Si(OH)₄, the binding capacity of pools is high, and a nonsaturable surge occurs (Fig. 8C). Higher V_max values measured at 2, 10, and 30 min versus those at 2 and 3 h (Fig. 1; Table II) suggests that over time, nonsaturable uptake transitions to saturable uptake because equilibrium is achieved between the capacity of binding components and their delivery rate of Si to the cell wall (Fig. 8D); thus, the rate of uptake becomes controlled by the rate of cell wall silica incorporation (i.e. internally controlled uptake). In extensively (24 h) Si-starved cultures of T. pseudonana (Fig. 8, E and F), pools also increase rapidly upon Si(OH)₄ addition but not to the extent seen in Figure 5A, and rather than decrease over time, they gradually increase (Hildebrand et al., 2007). Pool levels are minimal after prolonged Si starvation (Martin-Jézéquel et al., 2000); thus, we propose that levels of soluble Si-binding component decrease (Fig. 8E), and the diminished intracellular capacity results in saturable kinetics immediately upon addition of Si(OH)₄. Pool levels then gradually increase (Hildebrand et al., 2007) to maintain equilibrium with the increasing demand for silica incorporation, and presumably a concurrent increase in the amount of Si-binding component occurs.

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   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.

Transition from Short-Term Nonsaturable to Long-Term Saturable Uptake Kinetics Occurred in All Diatom Species Tested

All species tested followed a similar trend toward saturation as T. pseudonana. The rate at which saturability was achieved varied depending on the species (Fig. 6B), with the exception of T. weissflogii, in which uptake approached saturability but was not achieved. At least three variables could come into play regarding how fast saturability is achieved: the rate of uptake (how fast the pools are replenished), the rate of silica incorporation (how fast the pools are depleted), and the rate of change in capacity of the pools (i.e. the amount of available binding component). A simplistic way of determining the effect of pools on saturability is to compare the ratio of cell wall silica to pools (Fig. 7), with the assumption that a higher ratio means pools would be depleted more rapidly and saturation would occur faster. Analyzing data for uptake and rate of saturation (Fig. 6) and cell wall silica to pools (Fig. 7) did not reveal any clear trends in this regard; however, three interesting observations could be made. First, the lack of saturability in T. weissflogii could relate to this diatom's ability to maintain an entire cell wall worth of Si in intracellular pools (Binder and Chisholm, 1980; Brzezinski and Conley, 1994). Second, in comparing the two N. pelliculosa species, which have similar cell sizes and shapes, N. pelliculosa FW had a 4.3-fold higher cell wall to pools ratio and a 2.9-fold faster rate of saturation, consistent with the pools/incorporation equilibration hypothesis. Third, there is a general correlation between cell size (data not shown) and rate to achieve saturation (Fig. 6B), suggesting cell volume or geometry may have an effect. Cell size has previously been shown to influence V_max, where cells with higher surface to volume ratios have a lower V_max (Leynaert et al., 2004). Interestingly, C. gracilis, which has 80% of its cell wall silica in the form of long and narrow spines (Rogerson et al., 1986), had one of the fastest saturation times (Fig. 5B), even though its cell wall to pools ratio was near the average of the species examined.

Environmental Relevance

Concentrations of Si(OH)₄ in most of the ocean's photic zone average 10 µmol L^–1 (Tréguer et al., 1995). Based on our data, Si(OH)₄ uptake by field assemblages would mostly be directly controlled by SITs, which would minimize the associated costs of excess surge uptake and efflux. Intracellular pools should be in equilibrium with the needs for cell wall silica; thus, uptake rates could relate to incorporation rates. In coastal environments or upwelling zones, conditions favoring surge uptake could occur; for example, cells exposed to high levels of Si(OH)₄ would have a higher intracellular pool capacity, allowing surge uptake to occur more frequently. Certain ocean, and many freshwater, locations have Si(OH)₄ levels in the realm of diffusion mediated uptake. Antarctic ocean waters can contain as high as 100 µmol L^–1 in the winter (Tréguer et al., 1995); thus, diffusion-mediated uptake could play an important role in regulating diatom growth. In addition, diatoms once continuously experienced near saturating levels of Si (approximately 2 mM; Holland, 1984), raising the intriguing possibility that SITs initially evolved as Si effluxers and then optimized uptake ability as oceanic Si levels dropped.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Culture Conditions

Thalassiosira pseudonana Hasle et Heimdale clone 3H CCMP1335 (Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences), Thalassiosira weissflogii (Grunow) Fryxell et Hasle CCMP1336, Cylindrotheca fusiformis Reimann et Lewin CCMP343, Navicula pelliculosa (Bréb.) Hilse UTEX 668 (Culture Collection of Algae at the University of Texas at Austin; a freshwater strain, referred to as N. pelliculosa FW in text), N. pelliculosa (Breb. et Kuetzing) Hilse CCMP543 (a marine strain, referred to as N. pelliculosa M in text), Phaeodactylum tricornutum Bohlin CCMP1327, Chaetoceros gracilis Schütt UTEX LB2658, and Nitzschia alba Lewin and Lewin CCMP2426 were grown in batch culture under continuous illumination with cool-white fluorescent lights at approximately 150 µmol quanta m^–2 s^–1 at 18°C to 20°C. N. pelliculosa FW was grown in freshwater tryptone medium (Reimann et al., 1966). All other diatom species were grown in artificial seawater (ASW) medium (Darley and Volcani, 1969) with biotin and vitamin B12 added to 1 ng L^–1 or f/2 medium made with 0.2 µm filtered and autoclaved local seawater (Guillard and Ryther, 1962; Guillard, 1975).

Si(OH)₄ Uptake Kinetic Measurements

Si(OH)₄ uptake rates vary at different stages of the cell cycle (Brzezinski, 1992). In addition, Si requirements at different cell cycle stages vary depending on the species (Brzezinski et al., 1990; Martin-Jézéquel et al., 2000). Thus, to avoid possible cell cycle and cell phasing effects, which are difficult to controllably reproduce, exponentially growing cultures in continuous light were used in all measurements. Preliminary measurements were done to determine the optimal cell concentration for each species that would result in counts significantly above background. This was 2.5 x 10⁵ cells mL^–1 for most species with the exception of P. tricornutum and N. pelliculosa FW, which required 2.0 x 10⁶ cells mL^–1. Si(OH)₄ uptake was measured using the radiotracer analog of silicic acid, ⁶⁸Ge(OH)₄, because it is available in high specific activity and carrier-free form (enabling short-term uptake measurements), has a relatively long half-life of 282 d, and has well-established protocols for measuring silicic acid transport in diatoms (Azam, 1974; Azam et al., 1974; Sullivan, 1976, 1977). Stock solutions containing 1.0 µCi mL^–1 of ⁶⁸Ge(OH)₄ and 10-fold concentrations of freshly made sodium silicate in 3.5% NaCl (MilliQ-treated water for N. pelliculosa FW) were prepared, and for a given concentration of silicate, 100 µL of ⁶⁸Ge:silicate stock per 1 mL of cells was assayed. For most experiments, 1, 2, 4, 8, 10, 15, 30, and 100 µmol L^–1 final silicate concentrations were used, with duplicates being done for 8 to 30 µmol L^–1. Cells were harvested by centrifugation at 3,000g for 5 min, then washed and resuspended in Si-free medium. For all experiments, with the exception of Figure 4B, cells were maintained in Si-free medium for 5 to 10 min (referred to as short-term Si starvation) prior to measuring Si(OH)₄ uptake. Long-term Si starvation was done for 24 h prior to measurements (Fig. 4B). After the addition of various silicate concentrations, cells were incubated in the light and aliquots removed at different incubation times, vacuum filtered onto a Millipore Isopore 1.2-µm RTTP membrane, and washed with 5 mL of 3.5% NaCl (MilliQ-treated water for N. pelliculosa FW). Background was minimal with this wash treatment. Short-term uptake refers to uptake measured after 2 min; long-term uptake refers to uptake measured after 1 to 3 h. For measurement of background, 1 mL of cells was added to separate tubes containing 100 µL of ⁶⁸Ge:silicate and immediately pipetted onto a Millipore membrane and washed. This manipulation took 8 to 10 s, so cellular binding and some uptake may have occurred during this time, but because this would be minimal and a consistent procedure was used in all experiments, subtraction of 0 min from the longer time points was used to calculate net uptake for those time points. Filters were counted either dry using a LKB Wallac 1282 Compugamma CS Universal gamma counter (Perkin-Elmer) or in 10 mL of water by Cherenkov counting in a Beckmann LSC6000TA scintillation counter (Beckman Coulter). Counts per minute were converted to femtomoles per cell per hour, taking into account counting efficiency, dilution effects, and radioactive decay. Data obtained using this method are referred to as Si(OH)₄ uptake. Data were analyzed using the software program GraphPad Prism 4. Uptake rates were expressed in hours even when short-term uptake was measured to more easily compare data between experiments and with past studies.

Inhibition of Uptake by Ge(OH)₄

Unlabeled Ge(OH)₄ was used as a competitive inhibitor of Si(OH)₄ uptake (Azam et al., 1974). Short-term (2 min) Si(OH)₄ uptake was measured in T. pseudonana as described above except that increasing concentrations of a constant ratio of Si(OH)₄:Ge(OH)₄ (1:0.33) were added.

Effect of Zinc Chelators on Si Uptake

A control experiment was done to test the effect of the membrane-impermeable divalent cation chelator Na₂EDTA and membrane-permeable zinc-specific chelator TPEN (Hitomi et al., 2001) on growth of T. pseudonana. A stock solution of 25 mmol L^–1 Na₂EDTA was made in water and of TPEN in 100% dimethyl sulfoxide (DMSO). Five-milliliter cultures of T. pseudonana were established with 1 x 10⁵ cells mL^–1 in the presence of Na₂EDTA and TPEN concentrations ranging from 0 to 1 mmol L^–1. Culture cell density was monitored after 8 d. For both chelators, no growth occurred above a concentration of 25 µmol L^–1, and equivalent volumes of DMSO alone did not inhibit growth. To confirm the specificity of TPEN for zinc, cultures were established at 2 x 10⁵ cells mL^–1 in the presence of 50 µmol L^–1 TPEN and increasing concentrations (50, 100, or 200 µmol L^–1) of CaCl₂, FeSO₄, or ZnCl₂. After 4 d, cell density was determined. To monitor the effect of the chelators on Si(OH)₄ uptake, exponentially growing cultures of T. pseudonana were harvested by centrifugation and washed in Si-free ASW. Cells were resuspended in Si-free ASW at a density of 2.5 x 10⁵ mL^–1. Chelators were added to separate tubes at a final concentration of 50 µmol L^–1. Control tubes had no addition or addition of an equivalent amoun t of DMSO as in the TPEN addition. These were allowed to incubate for 5 min after which 15 µmol L^{–1 68}Ge:Si(OH)₄ was added. Uptake was measured after 30 min as already described, with a 0-min time point used to calculate background.

Measurements of Cell Wall Silica and Intracellular Soluble Si Pools

Aliquots of cells (approximately 1.3 x 10⁷) during exponential growth were harvested in 14-mL Falcon 1029 polypropylene tubes and pelleted at 16,500g in an HB-4 rotor (Sorvall, Thermo Electron) for 4 min. Cells were washed in Si-free medium, repelleted, and stored at –20°C. Intracellular soluble Si pool levels were measured using the boiling water method of Sullivan (1979). Frozen cells were resuspended in 1 mL of MilliQ-treated water, boiled for 10 min, cooled, and then centrifuged for 5 min at 16,500g in an HB-4 rotor. Triplicate samples of supernatant were assayed for intracellular Si. Cell wall silica was determined by resuspending the boiled cell pellet in 4 mL of 0.5 N NaOH (made from solid NaOH dissolved in MilliQ water) and placing the tube in a boiling water bath for 15 min. After cooling, samples were neutralized with 2 mL of 1 N HCl and then centrifuged for 5 min at 16,500g in an HB-4 rotor. Triplicate samples of supernatant were assayed to measure cell wall silica. Silicic acid concentrations were measured using the silicomolybdate assay (Strickland and Parsons, 1968). The standard was sodium hexafluorosilicate (Sigma), used in a range of 0 to 50 µmol L^–1.

	ACKNOWLEDGMENTS

 
We thank Michael I. Latz and Bradley M. Tebo for use of lab space and equipment. We appreciate both encouraging words and helpful technical advice from Mark A. Brzezinski. We also thank Assaf Vardi for comments and suggestions on the manuscript and Adam B. Kustka for discussions about zinc. N. pelliculosa (Breb. et Kuetzing) Hilse CCMP543 was provided as part of a collaborative project with S. Hazelaar and W.W.C. Gieskes.

Received August 8, 2007; accepted December 20, 2007; published December 27, 2007.

	FOOTNOTES

 
¹ This work was supported by the Air Force Office of Scientific Research Multidisciplinary University Research Initiative (grant no. RF00965521 to M.H.).

² These authors contributed equally to the article.

³ Present address: Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kimberlee Thamatrakoln (thamat{at}marine.rutgers.edu).

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.107094

^* Corresponding author; e-mail thamat{at}marine.rutgers.edu.

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