Phosphorus-31 nuclear magnetic resonance studies of intracellular pH,phosphate compartmentation and phosphate transport in yeasts

³¹P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All ³¹P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by ³¹P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.Non-Standard Abbreviations NMR nuclear magnetic resonance - ppm parts per million - PP polyphosphate - P_i,c cytoplasmic inorganic phosphate - P_i,v vacuolar inorganic phosphate - pH_in,c cytoplasmic pH - pH_in,v vacuolar pH - FCCP carbonyl p-trifluoromethoxyphenylhydrazone     

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      

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Observations on the subcellular distribution of the ammonium ion in maize root tissue using in-vivo 14N-nuclear magnetic resonance spectroscopy

We show that the pH dependence of the base-catalysed exchange rate of the ammonium ion provides a basis for discriminating between the cytoplasmic and vacuolar pools of ammonium in plant tissues. In vivo, ¹⁴N-nuclear magnetic resonance spectra were recorded with and without ¹H-decoupling and information on the subcellular distribution of NH ₄ ⁺ was obtained from a lineshape analysis of the ¹H-coupled spectrum. We applied this method to maize (Zea mays L.) root tissues and found that: (i), the cytoplasmic ammonium concentration was low, which was in accord with the large activity of glutamine synthetase present in the roots; and (ii), inhibition of glutamine synthetase with methionine sulphoximine increased the cytoplasmic ammonium concentration, and led to the appearance of ammonium in the xylem sap.Abbreviations GS glutamine synthetase - MSO l-methionine sulphoximine - NMR nuclear magnetic resonance - Pi inorganic phosphate On secondment to the Department of Plant Sciences, University of Oxford.We acknowledge the financial support of the Agricultural and Food Research Council. R.B. Lee also thanks the Department of Plant Sciences, University of Oxford, for hospitality.     

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.     

31P nuclear magnetic resonance study of growth and dimorphic transition in Candida albicans

A 31P NMR study of the fungal pathogen Candida albicans was carried out. Yeast-form cells at different phases of growth, as well as germ tubes and hyphae were examined. In all cases, the NMR spectra showed well separated resonance peaks arising from phosphorus-containing metabolites, the most prominent being attributable to inorganic phosphate (Pi) polyphosphates, sugar phosphates and mononucleotides, NAD, ADP and ATP. Relevant signals were also detected in the phosphodiester region. The intensity of most signals, as measured relative to that of Pi, was clearly modulated both at the different phases of growth and during yeast-to-mycelium conversion, suggesting significant changes in the intracellular concentration of the corresponding metabolites. In particular, the intensity of the polyphosphate signal was high in exponentially growing, yeast-form cells, then progressively declined in the stationary phase, was very low in germ tubes and, finally, undetectable in hyphae. NMR spectral analysis of the Pi region showed that from early-stationary phase, Pi was present in two different cellular compartments, probably corresponding to the cytoplasm and the vacuole. From the chemical shift of Pi, the pH values of these two compartments could be evaluated. The cytoplasmic pH was generally slightly lower than neutrality (6.7-6.8), whereas the vacuolar pH was always markedly more acidic.     

P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using ³¹P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (≤ 0.20 millimolar), the hexose monophosphate and cytoplasmic phosphate peaks declined earlier in reproductive development than when phosphate was present in optimal amounts. Under low phosphate concentrations (0.05 millimolar) cytoplasmic phosphate was greatly reduced. Carbon metabolism was coincidently disrupted under low phosphate nutrition as shown by the appearance of large, prominent starch grains in the leaves. Cytoplasmic phosphate, and leaf carbon metabolism dependent on it, are buffered by vacuolar phosphate until late stages of reproductive growth.     

Role of cytoplasmic inorganic phosphate in light-induced activation of H+-pumps in the plasma membrane and tonoplast of Chara corallina

A unique variant strain of Chara corallina, which contains little inorganic phosphate in the vacuole ([Pi]_v) was isolated. The level of cytoplasmic inorganic phosphate ([Pi]_c) in these cells was the same as that in normal cells. Using these unique cells, we studied the change in [Pi]_c and the effect of Pi on the activities of electrogenic H⁺-pumps associated with the plasma membrane and tonoplast. Upon illumination, the plasma membrane of C. corallina became hyperpolarized by 15 mV, the pH of the vacuolar sap decreased by 0.5 unit, and [Pi]_c decreased by 30% with a similar time course. The activities of the electrogenic H ⁺-pump in the plasma membrane and the ATP and PPi-dependent H⁺-transport in the tonoplast were noncompetitively inhibited by Pi with K_i values of, in the order given, 21.3 mM, 22.1 mM and 37.7 mM. From the kinetics study we calculated that the electrogenic H⁺-pump in the plasma membrane and the ATP and PPi-dependent H⁺ transport in the tonoplast were activated by, again in this order, 13%, 13% and 9%, in accordance with the decrease in [Pi]_c. We propose that the change in [Pi]_c is one of the regulators of photosynthesis-mediated activation of the H⁺-pumps in the plasma membrane and the tonoplast in C. corallina upon illumination.     

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium

³¹P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn²⁺ and phosphate from the extracellular medium and on providing the O₂ supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose-6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied.     

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     

Characterization of the 31P NMR spectrum of Manduca sexta and effects of antimetabolites

High resolution ³¹P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta. Conditions for in vivo analysis under non-saturating conditions are described. The ³¹P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined.Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7.The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the ³¹P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta.     

Subcellular Distribution of Inorganic Phosphate, and Levels of Nucleoside Triphosphate, in Mature Maize Roots at Low External Phosphate Concentrations: Measurements with 31P-NMR

Maize plants were grown in nutrient solution without phosphate,or in which inorganic phosphate (Pi) was maintained at nearlyconstant concentrations of 1 µM, 10µM or 0·5mM. In vivo ³¹P-NMR measurements showed that there was no discernibledifference in the cytoplasmic Pi content (µmol cm^–3root volume) of the mature roots of plants exposed to 1 µM,10µM or 0·5 mM external phosphate for up to 12d. However, the vacuolar Pi content of the mature roots variedabout 10-fold between these three groups. The cytoplasmic Pi content of roots receiving no external phosphatedecreased significantly after about 7 d total growth, and atabout this time the vacuolar pool of Pi became too small foraccurate measurement. The presence of 1 µM Pi in the nutrientsolution completely prevented this decline in cytoplasmic Pi,and there was some evidence that it also raised the Pi contentof the root vacuoles above the almost undetectable level foundin the totally P-starved roots. During the first 7–9 d of growth, the nucleoside triphosphatecontent of the mature roots was unaffected by the concentrationof phosphate in the nutrient solution. The results highlight the close control of cytoplasmic concentrationsof certain important phosphorus metabolites in roots growingin soil of normal agricultural fertility. Key words: Vacuole, cytoplasm, intracellular compartmentation, NTP, P-nutrition     

Phosphorus-31 NMR Studies of Cell Wall-Associated Calcium-Phosphates in Ulva lactuca

Phosphate concentrations in the range 0.1 to 2.0 millimolar induced the formation of extracellular amorphous calcium-phosphates in the cell wall of the marine macro algae Ulva lactuca when they were cultivated in light in seawater at 20°C. A broad resonance representing these compounds as well as resonances for extracellular orthophosphate and polyphosphates could be followed by ³¹P-nuclear magnetic resonance spectroscopy. The presence of the calcium-phosphate made the cells brittle and it inhibited the growth of the macro algae and caused mortality within 1 week. The formation of the calcium-phosphates was influenced by the external phosphate concentration, the extracellular pH and the nature and concentration of the external nitrogen source. Furthermore, no formation of these compounds was observed when Ulva lactuca was cultivated in the dark, at low temperatures (5°C) or in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The complex could be removed through washes with ethylenediaminetetraacetate; this treatment did not alter the intracellular pH or the orthophosphate and polyphosphate pools and it restored growth.     

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     

Comparing diatom and Alexandrium catenella/tamarense blooms in Thau lagoon: Importance of dissolved organic nitrogen in seasonally N-limited systems

Diatom blooms in Thau lagoon are always related to rain events leading to inputs of inorganic nutrients such as phosphate, ammonium and nitrate through the watershed with time lags of about 1 week. In contrast, blooms of Alexandrium catenella/tamarense can occur following periods of 3 weeks without precipitation and no significant input of conventional nutrients such as nitrate and phosphate. Field results also indicate a significant drop (from 22–25 to 15–16 μM over 3 days) in dissolved organic nitrogen (DON) at the bloom peak, as well as a significant inverse relationship between A. catenella/tamarense cell density and DON concentrations that is not apparent for diatom blooms. Such dinoflagellate blooms are also associated with elevated (6–9 μM) ammonium concentrations, a curious feature also observed by other investigators, possibly the results of ammonium excretion by this organism during urea or other organic nitrogen assimilation.The potential use of DON by this organism represents short cuts in the nitrogen cycle between plants and nutrients and requires a new model for phytoplankton growth that is different from the classical diatom bloom model. In contrast to such diatom blooms that are due to conventional (nitrate, phosphate) nutrient pulses, Alexandrium catenella/tamarense blooms on the monthly time scale are due to organic nutrient enrichment, a feature that allows net growth rates of about 1.3 d⁻¹, a value higher than that generally attributed to such organisms.     

Microelectrode and 133Cs nuclear magnetic resonance evidence for variable cytosolic and cytoplasmic nitrate pools in maize root tips

The effect of nitrate uptake on the subcellular distribution of tissue nitrate in 2–5 mm maize root tips was investigated by two complementary methods. First a novel in vivo analysis using ¹³³Cs nuclear magnetic resonance (NMR) was used to demonstrate changes in the cytoplasmic and vacuolar pools during caesium nitrate uptake. This method depended on interpreting the nitrate-induced changes in the positions of the cytoplasmic and vacuolar caesium signals. The assignment of the signals was confirmed by using in vivo³⁹K NMR to observe the displacement of cytoplasmic potassium into the vacuole during caesium uptake, and in vivo¹³³Cs NMR to observe the displacement of cytoplasmic caesium into the vacuole during potassium uptake. Secondly nitrate-selective microelectrodes were used to quantify the change in the cytosolic nitrate activity that occurred in the outermost cells of root tips under the same conditions. Both methods showed that the detected nitrate pool increased over a period of 8–10 h in the presence of 10 m m nitrate and it is concluded that the data provide support for the view that homeostasis in the cytosolic and cytoplasmic nitrate pools is not necessarily an invariant characteristic of root tips.     

Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo³¹P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.     

Metabolic changes of the Antarctic green alga Prasiola crispa subjected to water stress investigated by in vivo 31P NMR

The energy status and the phosphate metabolism of Prasiola crispduring and after desiccation stress was investigated by in vivo³¹P NMR. The effect of desiccation was simulated by additionof the nonionic osmoticum PEG 200 (polyethylene glycol). Photosynthesisand respiration were effectively inhibited under these conditions.The most notable changes in the in vivo ³¹P NMR spectra werean increase in the cytoplasmic inorganic phosphate signal afterPEG stress, a decrease in the polyphosphates and a lowfieldshift of the core polyphosphate signal followed by an appearanceof extracellular inorganic phosphate. Cytoplasmic pH remainedalmost constant during stress. After a return to control conditions,photosynthesis and respiration recovered within 4 h as wellas the concentrations of the phosphorus metabolites. An as yetunassigned phosphate signal increased in the phosphodiesterregion of the NMR spectra. Simultaneousty, the polyphosphatesignal recovered in intensity and chemical shift. It is suggestedthat phosphate metabolism and complexation of cations to polyphosphatesmay play an important role in the distinct desiccation toleranceof P. crispa. Key words: In vivo ³¹P NMR, Prasiola crispa, desiccation tolerance, polyphosphates     

<Superscript>31</Superscript>P NMR study of polyphosphate levels during different growth phases of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from ³¹P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22°C to 18°C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and β-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.     

Effects of aluminum on the release and-or immobilization of soluble phosphate in corn root tissue

The effects of aluminum ions on the generation of mobile inorganic phosphate (Pi) within the cells of excised maize (Zea mays L.) root tips were examined using ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy. When perfused with a solution containing 50 mM glucose and 0.1–5.0 mM Ca²⁺ at pH 4.0, 3–5-mm-long excised maize root tips from 3-d-old seedlings showed a significant (approx. 100%) increase in the amount of mobile Pi, (primarily vacuolar) over a period of 30 h. This increase was above that which can be accounted for by the hydrolysis of endogenous sugar phosphates and nucleotides. A change of the pH of the perfusion solution to 7.0 reduced the increase in Pi to approx. 50%. Omission of Ca²⁺ in the solution at pH 4.0 caused the mobile Pi to increase to about 170%. However, the presence of Al³⁺ or both Ca²⁺ and Al³⁺ in the solution resulted in a significant loss (35–50%) of mostly vacuolar Pi over the same period of time. When root tips containing up to 65% of newly released Pi, produced after 20 h perfusion, were exposed to Al³⁺, no additional increase in the level of the mobile-Pi signal area was noted. Exposure to Al³⁺ with Ca²⁺ and glucose under hypoxia at pH 4.0 resulted in a threefold decrease in intracellular Pi content after the root tips were returned to aerobic conditions. These results indicate that external pH plays an important role in the generation of mobile intracellular Pi and that the presence of both Ca²⁺ and Al³⁺ can independently suppress the production of this excess Pi and ultimately reduce the vacuolar Pi.Abbreviations and symbols NMR nuclear magnetic resonance - Pi morganic phosphate - UDPG uridine diphosphoglucose - chemical shift