Adaptive phosphate uptake behaviour of phytoplankton to environmental phosphate fluctuations

When phytoplankton growth in lakes is limited by the available phosphate, the external phosphate concentration fluctuates around a threshold value at which available energy is insufficient to drive phosphate incorporation into a polyphosphate pool. As a result, occasional increases in the external concentration are experienced by phytoplankton as a series of phosphate pulses. Based on [(32) P] phosphate uptake experiments with lake phytoplankton, we show that a community is able to process information about the experienced pattern of phosphate pulses via a complex regulation of the kinetic and energetic properties of cellular phosphate uptake systems. As a result, physiological adaptation to alterations of ambient phosphate concentration depends on the pattern of phosphate fluctuations to which the community had been exposed during its previous growth. In this process, the entire community exhibits coherent uptake behaviour with respect to a common threshold value. Thereby, different threshold values result from different antecedent pulse patterns, apparently unrestrained by the amount of previously stored phosphate. The coherent behaviour observed contradicts the basic assumptions of the competitive exclusion principle and provides an alternative perspective for explaining the paradoxical coexistence of many phytoplankton species.     

Bioenergetic characterization of transient state phosphate uptake by the cyanobacterium Anacystis nidulans

A force flow relationship based on nonequilibrium thermodynamics was derived to analyze the variable transient state phosphate uptake phenomena of cyanobacteria seen under different growth conditions and external phosphate concentrations. This relationship postulates the following basic properties of the uptake system: First, a threshold value exists, below which incorporation is energetically impossible. Second, threshold values are influenced by the activity of the phosphate uptake system, such that a decrease of the activity increases the threshold level. Third, near the thermodynamic equilibrium the uptake rate is linearly dependent on the free energy of polyphosphate formation and the pH-gradient at the thylakoid membrane. Experiments performed with Anacystis nidulans showed that phosphate uptake characteristics conformed to the properties predicted by the linear force-flow relationship. Linearity extented into regions far form thermodynamic equilibrium, e.g. to high phosphate concentrations, when algae were preconditioned to high phosphate levels. Under phosphate limited growth linearity was confined to a small concentration range, threshold values decreased below 10 nM, and the external concentration approached threshold. The data suggest that the uptake system responds to changes in the external phosphate concentration in the same way as sensory systems to input stimuli by amplifying signals and adapting to them.Abbreviations chl chlorophyll - H _e ⁺ , H _C ⁺ , H _T ⁺ protons in the external medium, the cytoplasmic and thylakoid space respectively - P_c phosphate in the cytoplasmic space - P_e phosphate in the external medium - P_n, P_n+1 polyphosphates - pH_T pH-gradient across the thylakoid membrane     

Adaptive changes in phosphate uptake by the fungus Neurospora crassa in response to phosphate supply.

The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 micronM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The Km of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the Km of the high-affinity system was independent of the phosphate concentration of the growth medium. The Vmax of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the Vmax of the low-affinity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .     

Short-term phosphate uptake kinetics in Zostera noltii Hornem: a comparison between excised leaves and sediment-rooted plants

Short-term phosphate uptake by excised leaves of Zostera noltii Hornem. as well as by leaves of sediment-rooted plants were characterized and compared in a kinetic framework. Time courses of phosphate disappearance were measured over a wide range of initial substrate concentrations. Phosphate uptake determined by this perturbation method did not follow Michaelis-Menten kinetics. Both excised leaves and sediment-rooted plants exhibited a biphasic uptake pattern as a function of phosphate concentration. However, rooted plants showed higher uptake rates and accumulated higher amounts of phosphate than excised leaves. The results point out the importance of the structural and functional coupling between shoots and underground parts during the nutrient foliar uptake processes. Our study also indicates that Zostera noltii leaves function as a phosphate sink in the water column.A second objective of this work is to compare the perturbation and the multiple flask methods in determining the uptake kinetic parameters. The obtained results support that both methods provide valuable and complementary information in determining the uptake rates.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots

A very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. The roots of these plants are potentially capable of absorbing P from the soil solution both directly through root epidermis and root hairs, and via the AM fungal pathway that delivers P to the root cortex. A large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Recent work has identified additional P transporters in several unrelated species that are strongly induced, sometimes specifically, in AM roots. The primary aim of the work described in this paper was to determine how mycorrhizal colonisation by different species of AM fungi influenced the expression of members of the Pht1 gene families in the cereals Hordeum vulgare (barley), Triticum aestivum (wheat) and Zea mays (maize). RT-PCR and in-situ hybridisation, showed that the transporters HORvu;Pht1;8 (AY187023), TRIae;Pht1;myc (AJ830009) and ZEAma;Pht1;6 (AJ830010), had increased expression in roots colonised by the AM fungi Glomus intraradices,Glomus sp. WFVAM23 and Scutellospora calospora. These findings add to the increasing body of evidence indicating that plants that form AM associations with members of the Glomeromycota have evolved phosphate transporters that are either specifically or preferentially involved in scavenging phosphate from the apoplast between intracellular AM structures and root cortical cells. Operation of mycorrhiza-inducible P transporters in the AM P uptake pathway appears, at least partially, to replace uptake via different P transporters located in root epidermis and root hairs. Electronic Supplementary Material Supplementary material is available for this article at     

Acyl phosphate and cyclic AMP inhibition of reactions of d-glyceraldehyde-3-phosphate dehydrogenase with aldehyde and acyl phosphate substrates: Multiple inhibition analysis

The effects of the inhibitors trimethylacetyl phosphate and cAMP have been determined in reactions catalyzed by d-glyceraldehyde-3-phosphate dehydrogenase. These inhibitors must influence the oxidation of aldehydes through substrate dependent co-operative conformational changes. Both trimethylacetyl phosphate and cAMP give sigmoidal $\text{1}\text{V}$ vs (I) plots in oxidation of glyceraldehyde 3-phosphate, but exert linear competitive effects on the acyl phosphatase site in acylation reactions of β-(2-furyl) acryloyl phosphate. The linear inhibition in the latter reactions indicates that one inhibitor molecule is bound per active site. Hydride transfer to NAD⁺ is the ratedetermining step in oxidation of benzaldehyde to an acylenzyme, as shown by the threefold decrease in V_max without change in K_m when 1-deuterobenzaldehyde is the substrate; it is very likely this step that is affected by acyl phosphate inhibitors. Plots of $\text{1}\text{V}$ vs cAMP concentration for oxidation of benzaldehyde at a series of trimethylacetyl phosphate concentrations are parallel at concentrations of acyl phosphate less than 0.00625 m, which demonstrates that binding of the inhibitors is mutually exclusive. However, at higher concentrations of trimethylacetyl phosphate, the slopes are affected, which shows that both inhibitors are then binding. Thus, the binding of high concentrations of acyl phosphate must result in a conformational change of the enzyme that permits binding of both inhibitors. A number of conformations with different kinetic properties are formed with the various substrate and inhibitor combinations. In reactions of muscle d-glyceraldehyde-3-phosphate dehydrogenase, binding of these inhibitors is best explained in terms of induced fit and a sequential model of conformational changes.     

Phosphate Uptake by Eukaryotic Algae in Cultures and by a Mixed Phytoplankton Population in a Lake: Analysis by a Force-Flow Relationship*

The phosphate uptake behaviour of monospecific cultures of green algae in the laboratory and of mixed phytoplankton populations in a mesotrophic lake has been analyzed with the aid of a force-flow relationship. This analysis yields two parameters:

• 1 A conductivity coefficient, that characterizes the activity of the phosphate uptake system.
• 2 An external threshold phosphate concentration, below which uptake of phosphate is excluded on energetic grounds.

When the phosphate concentration lies below the threshold value, the algae show an activation of the uptake system, reflected in an increase in the conductivity coefficient. Correspondingly, excess phosphate above the threshold value leads to a diminuation of the conductivity. Using this simple analysis, phosphate discharge into lake water may be readily monitored.     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.     

The bioenergetic coordination of a complex biological system is revealed by its adaptation to changing environmental conditions

The properties of the phosphate uptake system of the cyanobacterium Anacystis nidulans have been studied during the transition from a phosphate-deficient non-growing state to a non-deficient growing state. In the phosphate-deficient state the high affinity phosphate transport system in the cell membrane is extremely adaptive. As a result of these adaptive features the phosphate transport system cannot be described by determinate, fixed parameters, because the transport system is influenced by the measurement of the uptake process itself. When the growing state has been initiated by a persisting phosphate pulse, the transport system rapidly loses its adaptive features and can then be characterized by determinate parameters that remain unchanged for a long period of time, even if no uptake occurs in that time. Depending on the amount of phosphate stored during a pulse the cell makes a choice between slow or fast growth. In the latter case the light harvesting and energy converting machinery is completely reorganized before growth commences. Thereby the components of this machinery conform to each other and to the stable properties of the phosphate transport system. It is suggested that the mutual adjustment of these adaptive energy converting subunits is guided by attractors that function as the final cause for the development of the whole system.An application of this model to an analysis of the selforganization of aquatic ecosystems is discussed.     

Potassium removal accompanied by enhanced biological phosphate removal

The effects of potassium on excess uptake of phosphate in an aerobic-anaerobic activated sludge process were examined by the fill-and-draw procedure. The presence of sufficient potassium was necessary for excess uptake to occur. The contents of potassium and phosphate in the sludge at the end of each cycle of the process were correlated with each other by a non-linear equation, with some scattering. However, the sum of the molar ion valences of magnesium (Mg) and potassium (K), 2 Mg + K, was well correlated by a linear equation with the moles of P expressed as mmol/g-VSS, with a correlation coefficient of 0.993. When the potassium concentration was insufficient for the enhanced uptake of phosphate, its concentration in treated water was of the order of 0.1 mg/l. In the first anaerobic period phosphate was released into the liquid phase, but potassium was released after initial instantaneous removal, and in the successive aerobic period they were both taken up again.     

Solubilization of rock phosphate by rape

Rape and sunflower were compared with respect to their rock phosphate mobilizing capacities, cationanion balance and uptake of Ca and NO₃ at P-starvation. Rape was able to mobilize P from rock phosphate, whereas sunflower was not.When grown on a complete nutrient solution with NO₃ as the only nitrogen source, both species took up more nutrient anions than cations. Withholding phosphate from the nutrient solution did not change the uptake pattern of rape, but sunflower took up more nutrient cations than anions at P-starvation, due to a strong decline in NO₃ uptake. With both species, Ca uptake was not affected by phosphate in the nutrient solution.In split pot experiments, with rock phosphate supplied spatially separated from other nutrients, rape was still able to mobilize rock phosphate. A high Ca concentration had no effect on this capacity.The results indicated that in our experiments rock phosphate mobilization by rape was not associated with an excess of cation over anion uptake and neither with a high Ca uptake rate.     

Sodium-dependent transport of phosphate in LLC-PK1 cells

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK₁ cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na⁺-dependent, substrate-saturable process with an apparent K_m value for phosphate of 96 ± 15 μmol/1. Kinetic analysis of the effect of Na⁺ indicated that at (pH 7.4) two sodium ions are cotransported with one HPO₄^2? ion (Hill coefficient 1.5) with an apparent K_m value for sodium of 56 mmol/l. P_i uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 P_i uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK_i cells together with sodium (2 Na⁺ :1 HPO₄^2?) in an electroneutral manner down a favourable sodium gradient.     

Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa.

The kinetics of phosphate uptake by exponentially growing Neurospora crassa were studied to determine the nature of the differences in uptake activity associated with growth at different external phosphate concentrations. Conidia, grown in liquid medium containing either 10 mM or 50 micronM phosphate, were harvested, and their phosphate uptake ability was measured. Initial experiments, where uptake was examined over a narrow concentration range near that of the growth medium, indicated the presence of a low-affintiy (high Km) system in germlings from 50 micronM phosphate. Uptake by each system was energy dependent and sensitive to inhibitors of membrane function. No efflux of phosphate or phosphorus-containing compounds could be detected. When examined over a wide concentration range, uptake was consistent with the simultaneous operation of low- and high-affinity systems in both types of germlings. The Vmax estimates for the two systems were higher in germlings from 50 micronM phosphate than for the corresponding systems in germlings from 10 mM phosphate. The Km of the high-affinity system was the same in both types of germlings, whereas the Km of the low-affinity system in germlings from 10 mM phosphate was about three three times that of the system in germlings from 50 micronM phosphate.     

Growth optimization of the invasive cyanobacterium Cylindrospermopsis raciborskii in response to phosphate fluctuations

The role of ecophysiological traits in the success and expansion of the toxic cyanobacterium Cylindrospermopsis raciborskii is still under debate. One key factor appears to be the high physiological flexibility of this organism when obtaining limiting resources. Recent studies have found that filamentous bloom-forming cyanobacteria are able to optimize their growth by adjusting phosphate uptake during fluctuating nutrient conditions. We investigated the growth response of two phosphate-deficient C. raciborskii isolates (MVCC19 from Uruguay and CCMP1973 from USA) to short-term fluctuations in phosphate supply. These isolates were exposed to five phosphate concentrations which were provided in two supply modes: a single pulse (SingleP) versus the same amount divided in 10 pulses (TenP), with one pulse applied every 6 min. Morphological traits and changes in chlorophyll a and phycocyanin fluorescence were also evaluated. Growth rates of CCMP1973 and MVCC19 almost doubled and tripled, respectively, when exposed to multiple rather than single phosphate additions. Different growth rates were observed with the same total added resource, thus contradicting the classical model of dependence of growth rate on absolute external concentration. Phosphate-deficient C. raciborskii showed a remarkable physiological flexibility in adapting to phosphate availability on a timescale from minutes to hours. The TenP mode provided an extension of phosphate exposure time that allowed the energetic optimization of uptake and growth. The morphological plasticity of the species in response to phosphate supply mode was also shown by differences in trichome length and individual size. Our results are the first evidence of growth optimization of phosphate-deficient C. raciborskii to short-term nutrient fluctuations, revealing its physiological flexibility. This adaptive behaviour may help to explain the invasive success of this diazotrophic cyanobacterium in a wide range of aquatic ecosystems where phosphorus is frequently the limiting resource.     

Quantitative evaluation of the role of organic acid exudation in the mobilization of rock phosphate by rape

Phosphorus-deficient rape plants appear to acidify part of their rhizosphere by exuding malic and citric acid. A simulation model was used to evaluate the effect of measured exudation rates on phosphate uptake from Mali rock phosphate. The model used was one on nutrient uptake, extended to include both the effect of ion uptake and exudation on rhizosphere pH and the effect of rhizosphere pH on the solubilization of rock phosphate. Only the youngest zones of the root system were assumed to exude organic acids. The transport of protons released by organic acids was described by mass flow and diffusion. An experimentally determined relation was used describing pH and phosphate concentration in the soil solution as a function of total soil acid concentration. Model parameters were determined in experiments on organic acid exudation and on the uptake of phosphate by rape from a mixture of quartz sand and rock phosphate. Results based on simulation calculations indicated that the exudation rates measured in rape plants deficient in phosphorus can provide the roots with more phosphate than is actually taken up. Presence of root hairs enhanced the effect of organic acid exudation on calculated phosphate uptake. However, increase of root hair length without exudation as an alternative strategy to increase phosphate uptake from rock phosphate appeared to be less effective than exudation of organic acids. It was concluded that organic acid exudation is a highly effective strategy to increase phosphate uptake from rock phosphate, and that it unlikely that other rhizosphere processes play an important role in rock phosphate mobilization by rape.     

A comparison of two methods for measuring phosphate uptake by Monochrysis lutheri droop grown in continuous culture

Two methods for measuring phosphate uptake by phosphate-limited continuous cultures of Monochrysis lutheri Droop are found to yield comparable results. Data from both isotopic tracer (³³P) and disappearance experiments strongly support a Michaelis- Menten-type hyperbolic relationship between instantaneous uptake rate and ambient phosphate concentration. The data show no systematic linear trend in either the maximum uptake rate per cell or its associated half saturation constant with pre-conditioning steady state growth rate; a weak non-linear trend with pre-conditioning may, however, be present in the maximum uptake rate with an apparent maximum at intermediate growth rates.