Cellular Pi Concentration Research Articles

Proteomics reveals the significance of vacuole Pi transporter in the adaptability of Brassica napus to Pi deprivation.

Vacuolar Pi transporters (VPTs) have recently been identified as important regulators of cellular Pi status in Arabidopsis thaliana and Oryza sativa. In the oil crop Brassica napus, BnA09PHT5;1a and BnC09PHT5;1a are two homologs of AtPHT5;1, the vacuolar Pi influx transporter in Arabidopsis. Here, we show that Pi deficiency induces the transcription of both homologs of PHT5;1a genes in B. napus leaves. Brassica PHT5;1a double mutants (DM) had smaller shoots and higher cellular Pi concentrations than wild-type (WT, Westar 10), suggesting the potential role of BnPHT5;1a in modulating cellular Pi status in B. napus. A proteomic analysis was performed to estimate the role of BnPHT5;1a in Pi fluctuation. Results show that Pi deprivation disturbs the abundance of proteins in the physiological processes involved in carbohydrate metabolism, response to stimulus and stress in B. napus, while disruption of BnPHT5;1a genes may exacerbate these processes. Besides, the processes of cell redox homeostasis, lipid metabolic and proton transmembrane transport are supposed to be unbalanced in BnPHT5;1a DM under the -Pi condition. Noteworthy, disruption of BnPHT5;1a genes severely alters the abundance of proteins related to ATP biosynthesis, and proton/inorganic cation transmembrane under normal Pi condition, which might contribute to B. napus growth limitations. Additionally, seven new protein markers of Pi homeostasis are identified in B. napus. Taken together, this study characterizes the important regulatory role of BnPHT5;1a genes as vacuolar Pi influx transporters in Pi homeostasis in B. napus.

Identification of vacuolar phosphate influx transporters in Brassica napus.

Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homoeostasis in Arabidopsis thaliana and Oryza sativa under fluctuating external Pi supply, but the identity and involvement of VPTs in cellular Pi homoeostasis in Brassica napus is poorly understood. Here, we identified two vacuolar Pi influx transporters B. napus, BnA09PHT5;1b and BnCnPHT5;1b, and uncovered their necessity for cellular Pi homoeostasis through functional analysis. Both Brassica proteins are homologs of Arabidopsis AtPHT5;1 with a similar sequence, structure, tonoplast localization, and VPT activity. Brassica pht5;1b double mutants had smaller shoots and larger shoot cellular Pi concentrations than wild-type B. napus, which contrasts with a previous study of the Arabidopsis pht5;1 mutant, suggesting that PHT5;1-VPTs play different roles in cellular Pi homoeostasis in seedlings of B. napus and A. thaliana. Disruption of BnPHT5;1b genes also caused Pi toxicity in floral organs, reduced seed yield and impacted seed traits, consistent with the proposed role of AtPHT5;1 in floral Pi homoeostasis in Arabidopsis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved roles of BnPHT5;1bs in cellular Pi homoeostasis in this plant species.

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8–SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Live Imaging of Phosphate Levels in Arabidopsis Root Cells Expressing a FRET-Based Phosphate Sensor.

Phosphorous (P) is an essential macronutrient in all organisms serving various fundamental biological processes, and is one of the least available plant nutrients in the soil. The application of inorganic phosphate (Pi) fertilizers is frequent, but it has a high environmental and financial cost. Breeding crops for improved Pi use-efficiency is a promising plant-based solution to pursue a reduction of fertilizer dependency. Availability of tools for monitoring changes of plant cellular Pi concentration in real-time can contribute to advancing knowledge on the molecular basis of Pi transport and homeostasis in plants. Genetically encoded fluorescent sensors have provided new insight on cellular processes. Here, we show that two Pi Fluorescence Resonance Energy Transfer (FRET)-based sensors from the FLIPPi family, the low-affinity FLIPPi-30m and the high-affinity FLIPPi-4µ, can be expressed and analyzed in Arabidopsis thaliana with wild-type background. These FLIPPi sensors had not been tested in plants, but only in mammalian cell lines. We show FRET response and live imaging of Pi levels in seedling roots of Arabidopsis FLIPPi-30m and FLIPPi-4µ lines. Our results reinforce that sensors from the FLIPPi family are valuable tools for studying mechanisms of Pi transport and homeostasis in plants, and for research towards a more sustainable use of Pi fertilization.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis

The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP8, and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP8. Furthermore, we showed that InsP8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.

Molecular regulators of phosphate homeostasis in plants

An appropriate cellular phosphate (Pi) concentration is indispensable for essential physiological and biochemical processes. To maintain cellular Pi homeostasis, plants have developed a series of adaptive responses to facilitate external Pi acquisition and to limit Pi consumption and to adjust Pi recycling internally when the Pi supply is inadequate. Over the past decade, significant progress has been made toward understanding such regulation at the molecular level. In this review, the focus is on the molecular regulators that mediate cellular Pi concentrations. The regulators are introduced and organized according to their original identification procedures, by the forward genetic approach of mutant screening or by reverse genetic analysis. These genes are involved in Pi uptake, allocation or remobilization or are upstream regulators, such as transcriptional factors or signalling molecules. In the future, integration of current knowledge and exploration of new technology is expected to offer new insights into molecular mechanisms that maintain Pi homeostasis.

Induction of cell surface blebbing by increased cellular Pi concentration.

Cell surface blebbing is an early, reversible characteristic of anoxia described in several different cell types. Blebbing may lead to the formation of large terminal blebs, and ultimately cell death upon rupture of the membrane. In the present work, evidence is presented indicating that Pi may be the immediate mediator of cell surface blebbing: (1) cell surface blebbing can be induced in normoxic Ehrlich ascites tumour cells by a high extracellular concentration of Pp leading to an increase in the cellular Pi concentration; (2) anoxia induces sustained elevation of the cellular Pi concentration and (3) cell surface blebbing during anoxia is reversed upon reoxygenation, and the disappearance of blebbing depends on the decrease in cellular Pi concentration. The rate of disappearance of blebs may be enhanced by the simultaneous addition of adenine and inosine to the growth medium. This leads to a decrease in cellular Pi concentration and to an almost complete restoration of the cellular ATP concentration. It is suggested that Pi is an important mediator of anoxia induced cell damage.

Metabolic acidosis is a potent stimulus for cellular inorganic phosphate generation in uraemia.

1. During metabolic acidosis, significant fluxes of inorganic phosphate (Pi) may occur from cellular to extracellular fluid. In this study Pi was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma Pi (hyperphosphataemia) and cellular organic phosphate concentrations. 2. Before dialysis, the ratio of cellular to extracellular Pi concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2. 3. An increase in cellular Pi similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro. 4. The total Pi content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of Pi from 2,3-bisphosphoglycerate in the cells. This was accompanied by net efflux of Pi into plasma. 5. In addition, the increase in the steady-state cellular Pi concentration on adding a constant extracellular Pi load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane Pi gradient also contribute to the rise in cellular Pi observed at low pH. 6. At normal plasma Pi concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2. However, this inhibition was blocked when cellular Pi was increased by adding Pi to the plasma in vitro. 7. Metabolic acidosis is therefore a potent stimulus for Pi generation in erythrocytes, and this Pi may serve to stimulate glycolysis which is normally inhibited by low pH.

32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.

Cellular Pi concentration and Pi uptake do not always change in parallel in cultured fibroblasts and osteoblasts

Cellular Pi concentration and Pi uptake do not always change in parallel in cultured fibroblasts and osteoblasts

An index to quantify the apparent regulation of orthophosphate concentration in mammalian cells

We have shown previously that the concentration of orthophosphate (P,) in human erythrocytes and skeletal muscle is regulated in response to changes in the extracellular Pi concentration (Bevington et al., 1986). This is in marked contrast to earlier reports that P, simply distributes passively between mammalian cells and extracellular fluid (Deuticke, 1967; Tenenhouse & Scriver, 1975). To compare the extent of regulation of the cellular PI concentration between cell types or in a given cell type under a series of different conditions, some convenient method is needed to quantify the regulation. Wc therefore propose the following phosphate regulation index, p . If the relationship between the extracellular P, concentration (C, in mmol/l) and the cellular P, concentration (C,) is approximately linear, so that Cz = a + bC,, p is taken to be the ratio of the steady-state cellular P, concentration when C , = 0 to the cellular Pi concentration when C , = 1 mM, so that p = a / ( a + 6 ) . The value of 1 mM is chosen for the second extracellular P, concentration as this is approximately the concentration in normal human extracellular fluid. This index has some useful properties. In a cell which can regulate its P, concentration perfectly so that C', is constant as C, varies, p = 1; whereas in a cell in which P, is passively distributed so that C, is directly proportional to C,, p = 0. A furthcr advantage is that p is the ratio of two cellular P, concentrations, so that it is independent of the units in which the concentrations are expressed (e.g. mmol/l of cell water, or pmol/mg of protein). In Table 1 we summarize 10 published studies of the relationship betwecn C, and C, from which values of p can be derived. In seven of the 10 studies the relationship deviates markedly from that expected if P, distributes passively (because p 0.4). In -human erythrocytes, p seems to depend on the precise conditions under which it is measured.

Guard Cell Starch Biosynthesis Regulated by Effectors of ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase catalyzes the regulated step of starch bioynthesis in mesophyll chloroplasts. This enzyme is activated by a high ratio of the concentrations of 3-P-glycerate to inorganic phosphate (Pi) in light. In contrast, starch in guard cell chloroplasts is degraded when stomata open, which usually occurs in light. We have investigated the biochemical causes for this contrasting phenomenon.Vicia faba L. leaflets were sampled in darkness and after various periods of illumination. The samples were quick-frozen and freeze-dried. Guard cells and other cells were dissected out, weighed, and assayed for ADP-glucose pyrophosphorylase activity, 3-P-glycerate, and Pi. In the pyrophosphorolytic direction, ADP-glucose pyrophosphorylase specific activity in guard cells was 2.7 moles per kilogram protein per hour, which was comparable to the values obtained for palisade and spongy cells. The specific activity in epidermal cells was 4-fold lower. Under our assay conditions, the guard cell enzyme activity was 5-fold higher in the presence of 3-P-glycerate and 5-fold lower with Pi (i.e. similar to the results obtained with extracts of fresh leaflet). During three minutes of illumination, 3-P-glycerate concentration in palisade cells increased 2.5-fold to 10 millimoles per kilogram dry mass. The concentration of 3-P-glycerate in guard cells was 20-fold lower and unaffected by illumination. The concentration of Pi was approximately 17 millimoles per kilogram dry mass in palisade cells, but was 10-fold higher in guard cells. These overall cellular Pi concentrations were unaffected by illumination. We conclude that starch biosynthesis in guard cells is not activated by light because of the low and constant 3-P-glycerate concentration there. We interpret this last to be a consequence of the absence of the photosynthetic carbon reduction pathway in chloroplasts of these cells.

Phosphate concentration and transport in Ehrlich ascites tumor cells: effect of sodium.

The effects of extracellular Pi and Na+ on cellular Pi concentration and transport were studied. Steady-state Pi exchange flux was measured by 32P uptake in the presence and absence of Na+. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular Pi concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydrolysis of labile organic phosphate esters does not contribute to the measured intracellular pool of Pi. The Pi transport system exhibits an apparent Ks of 0.115 mM Pi and a maximal flux of 1.73 mmole min-1 (kg dry wt)-1. When incubated in a phosphate-buffered choline chloride medium (5 mM Pi) the intracellular Pi and the Pi influx fall by 65 and 88%, respectively. At 5 mM extracellular Pi, the Na+-dependent component of Pi transport fits Michaelis-Menten kinetics with the maximal flux equal to 2.46 mmole min-1 (kg dry wt)-1 and an apparent Ks of 35.4 mM Na+. In addition, a Na+-independent component of Pi transport, comprising about 12% of the total Pi flux, was identified. The data support the hypothesis that a Pi transport system, dependent on Na+, plays a principal role in the maintenance of intracellular Pi concentration.

The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis.

This paper is concerned with the control of glycolysis in nongrowing Streptococcus lactis 7962. Changes were measured in the concentrations of glycolytic intermediates, intracellular inorganic phosphate (Pi), and adenine nucleotides following addition of glucose to cells that were in a starved condition. We find that intracellular Pi is a major factor in the control of glycolysis. In starved cells, the intracellular Pi concentration is high, greater than 40 mM. The large phosphoenolpyruvate pool that exists in starved cells can be explained as a result of inhibition of pyruvate kinase by the high concentration of Pi. On the other hand, in cells that are metabolizing glucose at a steady state rate, the cellular Pi concentration is low and pyruvate kinase is active. Upon depletion of glucose from the medium, the metabolite concentrations return to the values originally found in the starved state. This glucose depletion raises the intracellular Pi which again leads to inhibition of pyruvate kinase and the consequent buildup of the P-enolpyruvate pool.