Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation      

Effects of confluence on phosphate transport capacity in cultured renal cell lines

The LLC-PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A-amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC-PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC-PK1 cells at confluence represents a halving in Vmax for Na-dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 microM). Suppression of phosphate transport in confluent monolayers of LLC-PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC-PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC-PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.     

Intracellular distribution of phosphate in cultured Humulus lupulus cells growing at elevated exogenous phosphate concentrations

The distribution of inorganic phosphate (Pi) between the cytoplasm and the vacuole of Humulus lupulus L. cells grown in suspension culture at different exogenous Pi levels was examined by 31-P nuclear magnetic resonance. In growing cells excess Pi accumulated in the vacuoles and the inhibitory effect of high exogenous Pi was not associated with a change in the cytoplasmic Pi level or with a change in the cytoplasmic pH.Abbreviations MES 2-(N-morpholino)ethanesulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate - ppm parts per million     

Phosphate exchange in the pit transport system in Escherichia coli.

The Pit system of phosphate transport in Escherichia coli catalyzes a rapid exchange between the external inorganic phosphate and internal phosphate pools, including some ester phosphates which are in rapid equilibrium with the internal Pi pool. Unlike net energized uptake, the Pi exchange proceeds in energy-depleted cells in the presence of uncouplers and is not accompanied by the movement of potassium ions. In the absence of externally added phosphate, the exit of Pi from the cells is insignificant. The apparent Km for external Pi in the exchange reaction is about 7 mM (2 orders of magnitude higher than that of energized uptake), but the maximal velocity is about the same. The exchange is temperature sensitive and is affected by thiol reagents. The combined observations suggest the operation of a facilitator which is part of the Pit system. The exchange is repressed in cells grown on glucose and other phosphotransferase system substrates, but not in cells grown on other carbohydrate sources. The repression can be reversed by the addition of cyclic AMP to the medium.     

Inorganic phosphate in Ehrlich ascites tumor cells and its distribution across the cell membrane

A regulatory function of the cell membrane in controlling the cytoplasmic level of Pi has been proposed, and in Ehrlich ascites tumor cells an active influx of primary phosphate has been reported in the literature. In the present study, Ehrlich cells were incubated at 1.5--50 mM extracellular Pi at pH 7.4 (Pi mainly secondary phosphate) and at pH 6.0 (mainly primary phosphate), and the measured cell Pi was compared with the value expected from a passive distribution of Pi. At a low extracellular Pi concentration the cell Pi was 3--6 mumol/g or even more. It is suggested that a major part of this cell Pi can be accounted for by enzymic release of Pi during the sampling procedure. If this interpretation is correct, the present results show that both ionic species of Pi are in electrochemical equilibrium across the cell membrane at steady state. Moreover, in vivo the concentration of free Pi in the cytosol will presumably be maintained at a steady-state level of about 0.4 mM, one order of magnitude below the directly measured values. This implies that the ratio [ATP]/[ADP][Pi] which is important in the regulation of energy metabolism, is higher than reported in the literature.     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

Imaging Cellular Inorganic Phosphate in Caenorhabditis elegans Using a Genetically Encoded FRET-Based Biosensor

Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

31P NMR Measurements of the Cytoplasmic and Vacuolar Pi Content of Mature Maize Roots: Relationships with Phosphorus Status and Phosphate Fluxes

The vacuolar and cytoplasmic inorganic phosphate (Pi) contentof the mature regions of maize roots was measured by a ³¹P NMRtechnique which used an external standard to avoid the needfor tissue extraction and which exploited the relatively rapidrelaxation of cytoplasmic Pi in order to improve the detectionof this pool in fully-vacuolated cells. In mature roots of maize growing with abundant external phosphate,the concentration of Pi in the cytoplasm was approximately 6.5mol m^–3. When these plants were deprived of external phosphate,the vacuolar Pi content of the roots decreased rapidly, butthe cytoplasmic Pi concentration initially remained constantand did not begin to decline until P-stress became severe. Calculationsshow that withdrawal of Pi from the vacuoles into the cytoplasmunder these conditions would be against an electrochemical gradient. During P-starvation, an increased capacity for Pi influx developed,preceding any detectable change in the cytoplasmic Pi contentof the roots. This response is considered in terms of paralleleffects on transport sites for phosphate at the plasmalemmaand at the tonoplast. Comparisons of simultaneous rates of influxand net uptake implied that phosphate efflux accounted for <10% of influx in plants of a steady or declining P-status. However,direct measurements of efflux suggested that this process maybe temporarily accelerated when plants are recovering from P-stress. Key words: P-nutrition, subcellular compartmentation     

Use of phosphate to avoid uranium toxicity in Arabidopsis thaliana leads to alterations of morphological and physiological responses regulated by phosphate availability

Uranium is an ubiquitous pollutant with known chemical and radiological toxicity, which is naturally present in the plant environment. Due to its high affinity for phosphate, insoluble uranium-phosphate precipitates are formed in soils as well as in contaminated plant cells. To date, consequences of such interactions on uranium toxicity and on phosphate availability and metabolism in plants are unknown. This study aims at evaluating in which extent uranium-phosphate interactions have an effect on physiological and molecular mechanisms involved in plant responses (i) to uranium contamination and (ii) to phosphate availability in Arabidopsis thaliana.Inorganic phosphate (Pi) supply in U-contaminated medium was shown to decrease U bioaccumulation and U toxic effects on plant biomass and root cell viability. Besides, U was shown to disturb plant responses to Pi availability. Indeed, in Pi-sufficient conditions, high U concentrations promoted the induction of phosphate starvation responses in plants. However, the most drastic effects have been observed in Pi-deficient conditions as U affected the following plant responses to Pi-starvation: root architecture modulation, phosphate acquisition and optimization of phosphate allocation. Indeed, despite the low Pi status of these plants, 2 μM U inhibited the primary root growth arrest normally triggered by low Pi. Moreover, Pi uptake and translocation to shoot were reduced. The root concentration of soluble inorganic phosphate decreased in Pi-starved plantlets contaminated with U, despite the enhancement of shoot-to-root remobilization of Pi. The observations of intracellular and apoplastic deposits of U and P in roots using electron microscopy (TEM-EDX) and secondary ion mass spectroscopy (NanoSIMS) provided evidence that Pi flux disturbance is a consequence of the use of Pi to immobilize U within roots.     

Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo³¹P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH ~6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N₂, the cytoplasmic Pi increased ~150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of ~10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H⁺-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.     

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.     

Extracellular inorganic phosphate regulates gibbon ape leukemia virus receptor-2/phosphate transporter mRNA expression in rat bone marrow stromal cells

In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells.     

Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae

Maintenance of ion homeostatic mechanisms is essential for living cells, including the budding yeast Saccharomyces cerevisiae. Whereas the impact of changes in phosphate metabolism on metal ion homeostasis has been recently examined, the inverse effect is still largely unexplored. We show here that depletion of potassium from the medium or alteration of diverse regulatory pathways controlling potassium uptake, such as the Trk potassium transporters or the Pma1 H⁺‐ATPase, triggers a response that mimics that of phosphate (Pi) deprivation, exemplified by accumulation of the high‐affinity Pi transporter Pho84. This response is mediated by and requires the integrity of the PHO signaling pathway. Removal of potassium from the medium does not alter the amount of total or free intracellular Pi, but is accompanied by decreased ATP and ADP levels and rapid depletion of cellular polyphosphates. Therefore, our data do not support the notion of Pi being the major signaling molecule triggering phosphate‐starvation responses. We also observe that cells with compromised potassium uptake cannot grow under limiting Pi conditions. The link between potassium and phosphate homeostasis reported here could explain the invasive phenotype, characteristic of nutrient deprivation, observed in potassium‐deficient yeast cells.     

Inorganic phosphate regulates CryIVA protoxin expression in Bacillus thuringiensis israelensis.

The role of nutritional factors during CryIVA protoxin expression in Bacillus thuringiensis israelensis (Bti) has been investigated. Inorganic phosphate (Pi) was found to stimulate 135 kD protoxin synthesis by Bti cells. There was a corresponding increase in the cryIVA specific mRNA in the presence of Pi. Inorganic phosphate inhibited HPr kinase but activated HPr phosphatase, the two enzymes responsible for regulating the concentration of phosphorylated HPr in the cell. Addition of protein phosphatase inhibitors NaF and calyculin A during resuspension resulted in the inhibition of toxin synthesis by Bti cells. Calyculin A inhibited HPr phosphatase activity in the in vitro assay also. The concentration of phosphorylated HPr was upregulated when the cells were resuspended in the presence of calyculin A, while the levels of the same were lowered in the presence of Pi, as determined by Western blotting the respective cells. The efficiency of sporulation of Bti was not affected when Pi was added alone or along with the phosphatase inhibitor calyculin A.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

Upregulation of vacuolar H(+)-translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures

The influence of phosphate (Pi) deprivation on the vacuolar H(+)-translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed. Pi starvation significantly elevated the ratios of PPi-:ATP-dependent H(+) translocation rate and H(+)-PPiase:H(+)-ATPase hydrolytic activities. These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi-starved cells. Immunoblotting indicated that Pi starvation also induced a two-fold increase in the amount of H(+)-PPiase protein, whereas the amount of H(+)-ATPase remained unchanged. It is proposed that H(+)-PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress.     

Phosphate accumulation in leaves of wheat seedlings measured in leaf cell protoplasts isolated after root or foliar treatment with orthophosphate

Leaf cell protoplasts were isolated from wheat seedlings ( Triticum aestivum L. cv. Urquie) after orthophosphate (Pi) treatment of the plant to determine the capacity for intracellular phosphate accumulation. Seedlings were treated with Pi concentrations near the phytotoxic level to maximize the Pi concentration in the leaf prior to protoplast isolation 1 day later. Both foliar and root treatment of seedlings with Pi increased the phosphate content of leaf protoplasts by approximately 20 μmol (mg chlorophyll)⁻¹ over Pi levels in untreated controls. Phosphate-loaded protoplasts from treated seedlings had similar photosynthetic rates and starch content but 50% more soluble reducing sugar than protoplasts from untreated seedlings. Protoplast dark respiration decreased after treatments which increased protoplast potassium content. The results suggest that similar amounts of Pi can be accumulated by leaf cells of wheat after foliar or root application of Pi to the seedling without hindering Pi-sensitive processes such as photosynthesis and starch synthesis.