Kinetics Of Pi Uptake Research Articles

Phosphorus Feast and Famine in Cyanobacteria: Is Luxury Uptake of the Nutrient Just a Consequence of Acclimation to Its Shortage?

To cope with fluctuating phosphorus (P) availability, cyanobacteria developed diverse acclimations, including luxury P uptake (LPU)—taking up P in excess of the current metabolic demand. LPU is underexplored, despite its importance for nutrient-driven rearrangements in aquatic ecosystems. We studied the LPU after the refeeding of P-deprived cyanobacterium Nostoc sp. PCC 7118 with inorganic phosphate (Pi), including the kinetics of Pi uptake, turnover of polyphosphate, cell ultrastructure, and gene expression. The P-deprived cells deployed acclimations to P shortage (reduction of photosynthetic apparatus and mobilization of cell P reserves). The P-starved cells capable of LPU exhibited a biphasic kinetic of the Pi uptake and polyphosphate formation. The first (fast) phase (1–2 h after Pi refeeding) occurred independently of light and temperature. It was accompanied by a transient accumulation of polyphosphate, still upregulated genes encoding high-affinity Pi transporters, and an ATP-dependent polyphosphate kinase. During the second (slow) phase, recovery from P starvation was accompanied by the downregulation of these genes. Our study revealed no specific acclimation to ample P conditions in Nostoc sp. PCC 7118. We conclude that the observed LPU phenomenon does not likely result from the activation of a mechanism specific for ample P conditions. On the contrary, it stems from slow disengagement of the low-P responses after the abrupt transition from low-P to ample P conditions.

Phosphorus starvation and luxury uptake in green microalgae revisited

Phosphorus (P) is central to storing and transferring energy and information in living cells, including those of microalgae. Many microalgal species dwelling in low P environments are naturally equipped to take up and store P whenever it becomes available through a complex phenomenon known as “luxury P uptake.” Its research is required for better understanding of the nutrient geochemical cycles in aquatic environments but also for biotechnological applications such as sequestration of nutrients from wastewater and production of algal fertilizers. Here, we report on our recent insights into luxury P uptake and polyphosphate formation originating from physiological, ultrastructural, and transcriptomic evidence. The cultures pre-starved of P and re-fed with inorganic phosphate (Pi) exhibited a bi-phasic kinetics of Pi uptake comprising fast (1–2 h after re-feeding) and slow (1–3 d after re-feeding) phases. The rate of Pi uptake in the fast phase was ca. 10 times higher than in the slow phase with an opposite trend shown for the cell division rate. The transient peak of polyphosphate accumulation was determined 2–4 h after re-feeding and coincided with the period of slow cell division and fast Pi uptake. In this phase, the microalgal cells reached the highest P content (up to 5% of dry cell weight). The P re-feeding also reversed the characteristic changes in cell lipids induced by P starvation, namely increase in the major membrane glycolipid (DGDG/MGDG) ratio and betaine lipids. These changes were reversed upon Pi re-feeding of the starved culture. Electron microscopy revealed the ordered organization of vacuolar polyphosphate indicative of the possible involvement of an enzyme (complex) in their synthesis. A candidate gene encoding a protein similar to the vacuolar transport chaperone (VTC) protein, featuring an expression pattern corresponding to polyphosphate accumulation, was revealed. Implications of the findings for efficient biocapture of phosphorus are discussed.

Uptake of phosphate by Synechocystis sp. PCC 6803 in dark conditions: Removal driving force and modeling

Rapid uptake of inorganic phosphate (Pi) by microalgae should occur through two processes operating in parallel: onto extracellular polymeric substances (EPS) and intracellular polymeric substances (IPS). Most previous studies focused only on overall Pi uptake and ignored the roles of EPS. We investigated the two-step removal of Pi by Synechocystis sp. PCC 6803 in dark conditions (i.e., without incorporation of Pi into newly synthesized biomass). We also developed a model to simulate both steps. Experimental results with Synechocystis confirmed that Pi in the bulk solution was removed by the two uptake mechanisms operating in parallel, but with different kinetics. All uptake rates decreased with time, and the Pi uptake rate by IPS was much higher than that by EPS at all times, but EPS had a larger maximum Pi-storage capacity -- 33–48 mgP/gCODEPS versus 15–17 mgP/gCODIPS. Synechocystis had a maximum Pi-storage capacity in the range of 22–28 mgP/g dry biomass. Protein in EPS and IPS played the key role for binding Pi, and biomass with higher protein content had greater Pi-storage capacity. Furthermore, biomass with low initial stored Pi had faster Pi-uptake kinetics, leading to more Pi removed from the bulk solution. This work lays the foundation for using microalgae as a means to remove Pi from polluted water and for understanding competition for Pi in microbial communities.

Possibilities of Optical Monitoring of Phosphorus Starvation in Suspensions of Microalga Chlorella vulgaris IPPAS C-1 (Chlorophyceae)

Studies of the impact of inorganic phosphorus (Pi), an important nutrient, on the growth and physiological parameters of single-celled algae are important for investigations of the dynamics of phytoplankton abundance and productivity in natural ecosystems as well as in industrial systems for the cultivation of microalgae. Difficulties in carrying out such studies are associated with the complex kinetics of Pi uptake by cells and the ability of microalgae to store phosphorus in their cells. This situation necessitates efficient methods for express monitoring of microalgal cultures, such as the methods based on the registration of optical properties of cells, in particular absorption and scattering of light and fluorescence of chlorophyll contained in the cells. Here, the results of monitoring the cultures of the chlorophyte Chlorella vulgaris IPPAS C-1 starving for phosphorus are described. It was found that both optical (light absorption in the bands of the key pigments—chlorophylls and carotenoids) and luminescent (variable fluorescence of chlorophyll) parameters closely reflect the culture condition. Registration of optical properties required correction for the contribution of light scattering to the overall extinction of light by microalgal cell suspensions. At the same time, the light scattering signal is an accurate measure of the total number of suspended particles in the suspension. However, it is difficult to monitor cultures containing a significant amount of light-scattering particles lacking photosynthetic pigments (such as heterotrophic bacteria). For such cultures, the use of variable fluorescence- based parameter Fv/Fm reflecting the maximum photochemical efficiency of the photosystem II is advisable.

Uptake of inorganic phosphorus by the aquatic plant Isoetes australis inhabiting oligotrophic vernal rock pools

The submerged aquatic freshwater macrophyte Isoetes australis S. Williams grows in rock pools situated in south-western Australia, an environment where dissolved inorganic phosphorus (Pi) availability possibly limits growth. In contrast to the two coexisting aquatic species, Glossostigma drummundii and Crassula natans, I. australis did not form relationships with mycorrhiza. Pi uptake kinetics were determined for I. australis in experiments using radioactive 33Pi. Roots had a higher Pi affinity (lowerKm) than leaves, but roots also had a lower Vmax, which is discussed in relation to the low ambient Pi concentrations. I. australis showed morphological adaptation which could relate to the low Pi environment by having approximately twice as much root tissue as leaf tissue (by dry mass), facilitating access to the higher P pools in the sediment compared with the shallow water column. A short-term translocation experiment revealed high amounts of Pi translocation internally in the plant which seemed to go from roots and oldest leaves to younger leaves. As a result of the high root to shoot ratio, high surface area, root uptake kinetics, and sediment Pi availability, roots accounted for 87% of plant Pi uptake and the green parts of the leaves for about the remaining 13%. As a result the estimated P budget for the rock pools showed that the surface water had a Pi turnover of about 1.5days, whereas the porewater Pi pool was renewed about 10 times per day to satisfy the P requirements of I. australis.

Phosphate uptake kinetics for four species of submerged freshwater macrophytes measured by a 33P phosphate radioisotope technique

Phosphate (Pi) uptake kinetics were determined in shoot and root tissues for four freshwater macrophyte species, Littorella uniflora, Potamogeton perfoliatus, Myriophyllum alterniflorum and Elodea canadensis, using a radioactive 33P phosphate technique. Collection of plant material in the oligotrophic softwater lake, Lake Hampen, Denmark, where Pi limits macrophyte growth, enabled us to characterize and compare the Pi uptake kinetics and competitive characteristics of the four species in a low level Pi environment. The maximum Pi uptake rates (Vmax), the half saturation constants (Km) together with the affinity at low Pi concentrations (Vmax/Km) were determined by fitting data to the Michaelis-Menten kinetics.L. uniflora showed the highest Vmax/Km in the root tissue and the lowest Km. M. alterniflorum showed the highest and E. canadensis and P. perfoliatus the lowest Vmax/Km in leaf tissue. M. alterniflorum had the highest Vmax and, as the only species, a higher Vmax in leaves than in roots. Surface area explained about half of Vmax in M. alterniflorum leaves. Roots were the dominant organ for Pi assimilation for all species at the Lake Hampen Pi concentrations. Km showed positive correlation to%P content in root tissue.The results indicate that at low lake water Pi concentrations L. uniflora is able to survive on the Pi pools in the sediment porewater. M. alterniflorum showed high affinity for Pi at both low and high Pi concentrations by both roots and shoots, and suggests that M. alterniflorum is a strong competitor at both low and high Pi concentrations.

Na+-stimulated phosphate uptake system in Synechocystis sp. PCC 6803 with Pst1 as a main transporter

BackgroundMost living cells uptake phosphate, an indispensable nutrient for growth from their natural environment. In Synechocystis sp. PCC 6803, the cells lack phosphate-inorganic transport (Pit) system but contain two phosphate-specific transport (Pst) systems, Pst1 and Pst2. We investigated the kinetics of Pi uptake of these two Pst systems by constructing the two mutants, ΔPst1 and ΔPst2, and comparing their kinetic properties with those of the wild-type cells under both Pi-sufficient and deficient conditions. The effects of pH and Na+ on the uptake of phosphate in Synechocystis were also studied.ResultsGrowth rates of the two mutants and wild type were similar either under phosphate-sufficient or deficient condition. The Km for phosphate uptake was 6.09 μM in wild type and this was reduced to 0.13 μM in ΔPst1 cells and 5.16 μM in the ΔPst2 strain. The Vmax values of 2.48, 0.22, and 2.17 μmol • (min • mg of chlorophyll a)-1 were obtained for wild type, the ΔPst1 and ΔPst2 strains, respectively. A monophasic phosphate uptake was observed in wild-type cells. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects.ConclusionThe data demonstrate that Pst2 system of Synechocystis has higher affinity toward phosphate with lower Vmax than Pst1 system. The Pst1 system had similar Km and Vmax values to those of the wild type suggesting that Pst1 is the main phosphate transporter in Synechocystis sp. PCC 6803. The Km of Pst1 of Synechocystis is closer to that of Pit system than to that of the Pst system of E. coli, suggesting that Synechocystis Pst1 is rather a medium/low affinity transporter whereas Pst2 is a high affinity transporter.

Dissolved inorganic and organic phosphorus uptake in Trichodesmium and the microbial community: The importance of phosphorus ester in the Sargasso Sea

The dynamics and relative importance of inorganic phosphate (Pi) and dissolved organic phosphorus (DOP) uptake were examined in Trichodesmium and the microbial community. Trichodesmium DOP and Pi uptake rates were also compared to literature values from several other taxonomically important groups in the Sargasso Sea. Uptake rates and uptake kinetics of 33Pi and DOP (using alpha‐labeled adenosine‐5′‐triphosphate 33P‐ATP as a model P ester compound) were assayed during two cruises. The in situ uptake rates suggest that the contribution of P ester to total P uptake can be greater than 25% for Trichodesmium, the microbial community, and key phytoplankton groups (Prochlorococcus, Synechococcus, picoeukaryotes, and nanoeukaryotes), depending on the location. Based on the kinetics of Pi uptake and P ester uptake, Trichodesmium is a poor competitor for Pi but a much better competitor for P esters as compared to the microbial community. Trichodesmium growth rates calculated from the Pi and P ester uptake parameters suggest that only a small fraction of the P ester pool needs to be bioavailable to allow reasonable growth rates. These data underscore the importance of P esters in supporting production by Trichodesmium in the Sargasso Sea, and provide the first in situ measurements of P ester uptake and uptake kinetics in this important N2‐fixing genus.

Mobilization and Acquisition of Sparingly Soluble P‐Sources by Brassica Cultivars under P‐Starved Environment II. Rhizospheric pH changes, Redesigned Root Architecture and Pi‐Uptake Kinetics

Non-mycorrhizal Brassica does not produce specialized root structures such as cluster or dauciform roots but is an effective user of P compared with other crops. In addition to P-uptake, utilization and remobilization activity, acquisition of orthophosphate (Pi) from extracellular sparingly P-sources or unavailable bound P-forms can be enhanced by biochemical rescue mechanisms such copious H(+)-efflux and/or carboxylates exudation into rhizosphere by roots via plasmalemma H(+) ATPase and anion channels triggered by P-starvation. To visualize the dissolution of sparingly soluble Ca-phosphate (Ca-P), newly formed Ca-P was suspended in agar containing other essential nutrients. With NH(4)(+) applied as the N source, the precipitate dissolved in the root vicinity can be ascribed to rhizosphere acidification, whereas no dissolution occurred with nitrate nutrition. To observe in situ rhizospheric pH changes, images were recorded after embedding the roots in agar containing bromocresol purple as a pH indicator. P-tolerant cultivar showed a greater decrease in pH than the sensitive cultivar in the culture media (the appearance of typical patterns of various colors of pH indicator in the root vicinity), and at stress P-level this acidification was more prominent. In experiment 2, low P-tolerant class-I cultivars (Oscar and Con-II) showed a greater decrease in solution media pH than low P-sensitive class-II (Gold Rush and RL-18) cultivars, and P-contents of the cultivars was inversely related to decrease in culture media pH. To elucidate P-stress-induced remodeling and redesigning in a root architectural system, cultivars were grown in rhizoboxes in experiment 3. The elongation rates of primary roots increased as P-supply increased, but the elongation rates of the branched zones of primary roots decreased. The length of the lateral roots and topological index values increased when cultivars were exposed to a P-stress environment. To elucidate Pi-uptake kinetics, parameters related to P influx: maximal transport rate (V(max)), the Michaelis-Menten constant (K(m)), and the external concentration when net uptake is zero (C(min)) were tested in experiment 4. Lower K(m) and C(min) values were better indicative of the P-uptake ability of the class-I cultivars, evidencing their adaptability to P-starved environmental cues. In experiment 5, class-I cultivars exuded two- to threefold more carboxylates than class-II cultivars under the P-stress environment. The amount and types of carboxylates exuded from the roots of P-starved plants differed from those of plants grown under P-sufficient conditions. Nevertheless, the exudation rate of both class-I and class-II cultivars decreased with time, and the highest exudation rate was found after the first 4 h of carboxylates collection. Higher P uptake by class-I cultivars was significantly related to the drop in root medium pH, which can be ascribed to H(+)-efflux from the roots supplied with sparingly soluble rock-P and Ca(3)(PO(4))(2). These classical rescue strategies provided the basis of P-solubilization and acquisition from sparingly soluble P-sources by Brassica cultivars to thrive in a typically stressful environment.

Thalassia testudinum phosphate uptake kinetics at low in situ concentrations using a 33P radioisotope technique

A new 33 P-tracer technique was used to define low-level phosphate (Pi) uptake kinetics for a dominant tropical seagrass, Thalassia testudinum. We established seagrass Pi uptake kinetics at high (western Bay) and low (eastern Bay) nutrient sites in a fine-grained carbonate lagoon of south Florida (Florida Bay), where P limitation of seagrass has been documented. Sediment Pi adsorption kinetics were also investigated to test whether carbonate sediments could sequester Pi to the threshold levels we established for T. testudinum, defined as S min or the physiological threshold value where Pi uptake is positive. At Pi levels characteristic of Florida Bay (#0.26 mmol L 21 ), leaf and root Pi uptake was linear with increasing levels of Pi, and similar affinities (a) were found for leaves and roots (0.12‐0.30 and 0.10‐0.20 mmol g dry weight 21 h 21 ). Smin for roots and leaves was extremely low (0.004‐0.009 mmol L 21 ) regardless of the P status of the site. While T. testudinum was able to take up Pi at nanomolar levels, the uptake rates were insufficient for plant requirements. Thus, sediment pools or transient fluxes of P to the water column must sustain high seagrass production rates in the Bay. Sediment adsorption‐desorption equilibrium for Pi was 10-fold lower in the eastern versus western Bay sites, meeting ,10% and .87% of the P demand for T. testudinum, respectively, a result that might account for the reported P limitation of seagrass biomass and production in eastern Florida Bay.

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas.

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.

PHOSPHORUS ACQUISITION IN FOUR CO‐EXISTING SPECIES FROM MONTANE GRASSLAND

SUMMARYPhosphorus acquisition was examined in four co‐existing species from montane grassland. The effect of inorganic phosphate (Pi) concentration on relative growth rate was studied and an attempt was made to determine whether inorganic or organic phosphates are the immediate sources of phosphorus for these plants using assays of Pi uptake kinetics and of root surface phosphatases. These data are also used to assess whether the availability of phosphorus might limit plant growth in this montane grassland.Potential uptake rates are unlikely to limit growth via Pi supply. There appears to be no preference for a specific phosphate source for growth with any species examined. Mycorrhizal infection does not appear to be a prerequisite for successful phosphate acquisition in these co‐existing species.