Utilization of limiting concentrations of ortho-phosphate and production of extracellular organic phosphates in cultures of marine diatoms

The utilization of ortho-phosphate by two coastal marine diatomspecies, Nitzschia closterium and Cyclotella cryptica, was studiedin batch cultures. The hypothesis was tested that thresholdconcentrations in the phosphate uptake determine the lower limitof environmental phosphate, permitting the existence of species.The turn-over time of residual medium phosphate in culturesis {small tilde}10 min, indicating a rapid equilibration ofconcentration dependent on uptake with leakage of ortho-phosphate.Increasing phosphate starvation in cultures diminished the residualortho-phosphate in the range of {small tilde}60–<2nmol l^–1, as measured radiochemically after elution onSephadex® G-10 gel. These concentrations encompass the rangeof limiting phosphate concentration in continuous cultures ofthe few microalgae, for which these concentrations are actuallymeasured. The diatoms excreted {small tilde}20–100 nmolI^–1 of organic phosphate. One dominating compound, probablyan unusual nucleotide, was incompletely or not resorbed underphosphate starvation. In contrast, Nitzschia closterium excretedunder ample phosphate supply a series of three related compounds,probably phospholipids, that were resorbed under depletion.The association of the organic phosphates with macromolecularexudates is interpreted, along with the other observations,as an indication for a hardly explored periplasmatic phosphatemetabolism in these algae. ³Dedicated to Prof. Dr. H.-A. von Stosch in honour of his 75thbirthday. ⁴This study was conducted at the University of Marburg undersupport of the Humboldt Foundation Publication no. 64 of theproject "Biological Research of the Eems-Dollard Estuary".     

Evaluation of Arsenate- and Vanadate-Associated Changes of Electrical Membrane Potential and Phosphate Transport in Lemna gibba G1

Interference of arsenate and vanadate with phosphate uptakein Lemna gibba L. was studied by measuring voltage changes and(³²P)phosphate uptake. Arsenate proved to be competitive withthe high- and low-affinity phosphate uptake system. It inducedtransient membrane potential changes of up to 120 mV which weresimilar to those induced by phosphate and indicated a cotransportmechanism with at least 2H+/H₂As. The amplitude of the transient arsenate-induced membrane depolarization wasstrongly influenced by phosphate starvation. A permanent membranedepolarization to the diffusion potential was achieved within2 to 6 h in P-starved plants. Thus, arsenate is indeed a stronglycompetitive physiological analogue of phosphate in higher plants. Vanadate was easily transported into L. gibba as concluded fromtransient E_m changes of up to 110 mV. Vanadate interfered onlyslightly and non-specifically with the two phosphate transportmechanisms. Like phosphate, vanadate uptake seems to be an H+-cotransportmechanism, both with similar optima at pH 6.0. Unlike phosphateuptake, vanadate-linked membrane depolarization was not affectedby high intracellular phosphate concentrations. P-starvationdid not enhance the weak long-term effect on E_m. Hence, vanadate,in contrast to arsenate, is not regarded as a physiologicalphosphate analogue. The distinct and rapid vanadate-inducedand permanent membrane depolarization of Avena sativa, Triticumaestivum and Glycine max leaves was not seen in Lemna nor inleaves of Gossypium hirsutum and Nicotiana tabacum. Plasmalemma-enrichedpreparations of L. gibba revealed, however, a high vanadate-sensitiveATPase activity (87%). As a possible explanation for these differencesit is suggested that the latter plant species have cytosolicvanadate-detoxifying properties, i.e. they can reduce vanadateto vanadyl ions, in contrast to the former group of plant species. Key words: Arsenate, vanadate, H⁺/solute cotransport, membrane potential, phosphate competition     

Characteristics of Aluminum-Phosphate-Adapted Carrot Cells: Uptake and Utilization of the Phosphate

To elucidate the mechanism responsible for the superior growthof a selected line of carrot cells (Daucus carota L. cv MS Yonsun)in medium that contained AIPO₄, kinetic studies of the uptakeof phosphate and the efficiency of utilization of phosphatewere performed with the selected cells and the wild-type cells.When the two cell lines were grown in a medium with adequatesoluble phosphate (2 mM), there was no difference between theirgrowth rates. Rates of increase in fresh weight as a functionof increasing concentration of phosphate in the medium werealso identical between the cell lines, indicating that the efficiencyof utilization of phosphate by the selected cell line was similarto that by the wild-type cells. However, rate of uptake of phosphateby the selected cells under phosphate limited conditions (20µMNaH₂PO₄ at pH 5.6) was about 5-fold higher than that by thewild-type cells. Apparent K_m values for the uptake of phosphatewere calculated to be 13.6 and 9.1 µM for the selectedand the wild-type cells, respectively. The V_max valuewas estimatedto be 88.8 nmol per g fresh weight per min for the selectedcells and 28.2 for thewild-type cells. Thus, the selected cellshas an enhanced system for uptake of phosphate wherebytherewas an increase in the rate of the uptake without any dramaticchange in the affinity for phosphateions. (Received September 21, 1991; Accepted December 25, 1991)     

Phosphate uptake by phosphorus-starved cells of the cyanobacterium Phormidium laminosum

Phosphorus(P)-starved cells of the cyanobacterium Phormidium laminosum have been investigated in relation to their phosphate uptake characteristics. P-deficient cells showed much higher phosphate uptake rates from ultrapure water supplemented with this anion than P-sufficient ones. After 9 days of starvation in P-free medium, the total cellular P content of P-deficient cells was approximately five times lower than that of cells grown in the presence of phosphate. Phosphate uptake by P-deficient cells occurred in both light and dark under aerobic conditions. In anaerobiosis, light was required for uptake, suggesting that the necessary energy could be derived from the respiratory electron transport chain. Phosphate uptake in P-deficient cells was sensitive to vanadate, suggesting the involvement of a plasma membrane ATPase.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Uptake of Inorganic Phosphate by Plastids Purified from the Roots of Pisum sativum L.

Pea root plastids are able to take up inorganic phosphate ina time-dependent reaction. The rate of uptake is reduced inthe presence of triose phosphates, though the kinetics of uptakedo not appear to conform to a simple Michaelis-Menten interpretation.Inorganic phosphate taken up by such preparations is lost fromthe plastids upon addition of triose phosphates. Unlike chloroplasts,root plastids do not appear to accumulate inorganic phosphateabove the concentration in the surrounding medium. Key words: Pisum, plastid, phosphate uptake     

Uptake of Inorganic Phosphate by Suspension-Cultured Tobacco Cells: Kinetics and Regulation by Pi Starvation

The kinetics of P_i uptake by phosphate-starved and non-starvedtobacco cells (Nicotiana tabacum BY-2) suspension culture wasinvestigated. The kinetic parameters of P_i uptake were determinedby computer simulation of the curve that represented the time-dependentloss of P_i from the culture medium. The uptake profile couldbe completely explained by assuming the existence of only onekind of Michaelis-Menten-type P_i-transport system with an affinityfor P_i (K_m) of about 2.5 µM (the lowest value reportedto date) in both P_i-starved and non-starved cells. No evidencewas obtained suggesting the existence of a "low-affinity" P_i-uptakesystem that has been postulated to exist in several other plantmaterials. The V_max for uptake of P_i by non-starved cells was12 nmol per minute per milliliter of packed cell. Phosphatestarvation increased the V_max more than 5-fold, while it hadno effect on the affinity for P_i. V_max began to increase (atan almost constant rate) just after loss of all P_i from theculture medium and it reached a maximum about 16 hours later.This induction process was completely prevented by the additionof cycloheximide to the culture medium. All these results suggestthat P_i starvation increases the synthesis of a phosphate-carriercomplex that is postulated to be involved in the P_i-uptake process. (Received August 12, 1994; Accepted December 26, 1994)     

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      

Phosphate Uptake and Transport by Roots and Stolons of Intact White Clover (Trifolium repens L.)

The extent to which phosphate can be absorbed directly fromthe outer medium by stolon internodes and contribute to thetotal accumulation of phosphate by intact plants of white clover(Trifolium repens L. cv. Blanca) was assessed in hydroponicexperiments in a controlled environment room. The uptake ofphosphate by intact roots or stolons was measured by sealinga segment (6-0 mm long) across a flow-cell in which ³²P-labellednutrient solution was circulated for 24 h, the rest of the rootsystem receiving unlabelled nutrient solution. The rate of uptakeof phosphate (µmol g^–1 d^–1 dry wt. basis)by roots was more than 300 times that by intact stolons. Pretreatmentof stolons by gentle abrasion to remove cuticle, so as to simulatethe condition of stolons in the field, increased the uptakeof phosphate 7-fold compared with that of intact stolons. However,the potential of stolons to contribute to the P status of whitedover in the field was calculated to be small (5%). When an incision was made through the hypodermal layer of stolons,the rate of phosphate uptake greatly increased, attaining 71%of that by root segments. This increase, which was greater athigher phosphate concentrations, indicates that the suberi.zedhypodermis constitutes a major barrier to the influx of phosphatein the stolon. After withholding phosphate for different time intervals, thesubsequent rate of phosphate uptake by roots was increased 2-3-foldafter 2 d phosphate deprivation and 3-4-fold after 6 d or 13d phosphate deprivation. A higher proportion of absorbed phosphatewas transported to shoots in phosphate-deprived plants. After1 d of uptake following restoration of the phosphate supply,the concentrations of labelled phosphate in shoots were greaterthan in control plants, although the concentrations of labelin roots was less. However, the rate of uptake of phosphateby stolons, following deprivation, was not significantly increased.These results suggest that the mechanism regulating the enhancedrate of phosphate loading into the xylem, initiated by a periodof phosphate deprivation, is specific to roots and is not inducedin stolons. The results are discussed in relation to the growth and acquisitionof phosphate by white clover in the field. Key words: Nutrient deficiency, phosphate, stolons, transport (ions), Trifolium repens     

The effect of acetate and succinate on polyphosphate formation and degradation in activated sludge,with particular reference to Acinetobacter calcoaceticus

Summary The effects of acetate and succinate were compared to the effect of phosphate starvation on the formation and degradation of polyphosphate in an Acinetobacter calcoaceticus isolate from a five-stage Bardenpho activated sludge plant and in mixed liquor from the same plant. Both acetate treatment and phosphate starvation result in significant phosphate release from the cells. Succinate treatment showed little difference from the control. A reduction in polyphosphate was observed simultaneously with the phosphate release. On resuspension of the treated samples in a complete medium, uptake of phosphate was observed. In the acetate-treated samples, this was significantly higher than in the phosphate starved samples. Polyphosphate formation was also significantly enhanced after treatment.     

Transient nitrate uptake and assimilation in Skeletonema costatum cultures subject to nitrate starvation under low irradiance

Batch cultures of the nitrate-grown marine diatom, Skeletonemacostatum, were grown in various levels of nitrate supply, fromfull sufficiency of nitrate to 96 h of nitrate starvation Allthe cultures were maintained at low light intensity (50 µEm^–2 s^–1) to simulate the light regime of naturalphytoplankton populations in turbid waters or at the edge ofthe photic zone. The response of S costatum cells to perturbationswith 10 µM nitrate after variable starvation periods wasstudied At the deficiency-starvation borderline, the specieswas able to both increase its uptake rate and store intracellularnitrate pools. Surge, or initially high, uptake is characteristicof this situation. After long starvation periods the cells neededto acclimate to a nitrate environment before being able to utilizeit The time required for this was proportional to the previousstarvation period. Time courses of nitrate uptake and reductionwere strongly non-linear. Nitrate excretion was high (up to3 µM) under intermediate (24 and 36 h) starvation periods.Differences in the rates and times of uptake, reduction andassimilation produced strong uncoupling of the three processesThe results suggest that there is an adaptation to nitrate utilizationunder low light, nitrate shortage and discontinuous, suddennitrate inputs.     

Phosphate uptake and utilization by bacteria and algae

Bacterial uptake of inorganic phosphate (closely investigated in Escherichia coli) is maintained by two different uptake systems. One (Pst system) is P_i-repressible and used in situations of phosphorus deficiency. The other system (Pit system) is constitutive. The Pit system also takes part in the phosphate exchange process where orthophosphate is continuously exchanged between the cell and the surrounding medium.Algal uptake mechanisms are less known. The uptake capacity increases during starvation but no clearly defined transport systems have been described. Uptake capacity seems to be regulated by internal phosphorus pools, e.g., polyphosphates. In mixed algal and bacterial populations, bacteria generally seem to be more efficient in utilizing low phosphate concentrations. The second half of this paper discusses how bacteria and algae can share limiting amounts of phosphate provided that the bacteria have pronouncedly higher affinity for phosphate. Part of the solution to this problem may be that bacteria are energy-limited rather than phosphate-limited and dependent on algal organic exudates for their energy supply.The possible phosphate exchange mechanism so convincingly demonstrated in Escherichia coli is here suggested to play a key role for the flux of phosphorus between bacteria and algae. Such a mechanism can also be used to explain the rapid phosphate exchange between the particulate and the dissolved phase which always occurs in short-term ³²P-uptake experiments in lake waters.     

Differences between Natural Populations of Trifolium repens L. in Response to Mineral Nutrients

The response to phosphate of samples of natural local populationsof T. repens, collected from soils of different phosphate contents,was determined over a wide range of phosphate levels in sandculture. A marked correlation (r = +0.96) was found betweenresponse of populations to phosphate at low phosphate levelsand the amount of phosphate in the native soil of the population;populations from soils high in phosphate showed larger reductionsin growth at low phosphate levels than did those from soilslow in phosphate. It is concluded that response to phosphateis one aspect of the physiological adaptation of the naturalpopulations to particular soil conditions. Populations alsodiffered in response to very high levels of phosphate, and inthe range of phosphate levels tolerated; the possible adaptivesignificance of this is discussed in relation to the phosphatebuffering effects of the native soils. Populations tolerantof low phosphate levels had higher concentrations of phosphorusin the shoot material, and a higher uptake of phosphorus perunit weight of roots, than did intolerant populations. The differencesbetween populations in response to phosphate are therefore believedto be due to difference in uptake rather than to differencein metabolic requirement of phosphate.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

A Simple and Inexpensive Design of Chemostat Enabling Steady-state Growth of Acer pseudoplatanus L. Cells under Phosphate-limiting Conditions

Operational and constructional details are given of a relativelysimple and inexpensive chemostat designed for the continuousculture of plant cells in suspension. This apparatus permitscontrol of the growth rate of sycamore, Acer pseudoplatanusL. cells in steady-state conditions. By alteration of the rateof input of medium different steady-state growth rates wereobtained over a wide range (mean doubling times from 182 h to36 h). In order to establish a growth-limiting nutrient thetime course of nutrient uptake in batch culture was measured.In batch culture the maximum growth obtained was proportionalto the initial concentration of phosphate when this was belowa concentration of 17 µg P per ml (as phosphate). It isalso shown in chemostat culture that the steady-state cell densityis proportional to the phosphate concentration in the mediumwhen this is below 17 µg P per ml (as phosphate). Phosphatewas therefore established to be the growth rate-limiting nutrientin chemostat culture at a concentration of 8•5 µgP per ml (as phosphate).     

Phosphorus uptake by resting cysts of the marine dinoflagellate Scrippsiella trochoidea

Resting cysts of the marine dinoflagellate Scrippsiella trochoideawere produced under phosphorus (P)-deficient conditions, separatedfrom vegatative cells, and incubated for 28 days in darknessat 4 and 20°C in P-enriched and P-deplete medium. The Pcontent of cysts incubated in the P-replete medium was significantlyhigher than that of cysts in P-deplete medium. As the P contentof the cysts increased through time, dissolved inorganic phosphatewas depleted in the medium. This decrease cannot be attributedto free-living bacterial uptake, since there was no correspondingincrease in bacterial particulate P. Disappearance of P fromthe medium can, therefore, only be explained by uptake associatedwith the cysts. This could be either direct cyst uptake, uptakeby bacteria closely associated with the cysts, or adsorptionof P on the cyst wall. Evidence is strongest that the cystsincorporated phosphate during the resting stages of dormancyand quiescence, despite the fact that these are periods of significantlyreduced metabolism. Accumulation of P during these benthic restingstages would increase the survival of newly excysted vegetativecells as they re-enter the water column after germination, providinga competitive advantage over other phytoplankton. Freshwaterand marine sediments provide a P-rich environment which mayserve as a potential nutrient pool for dinoflagellate restingcysts. Mobilization of nutrients to and from the sediments viacysts must now be evaluated to ascertain whether this couldbe a significant term in nutrient budgets.     

Uptake of Phosphate by White Clover: I. EVIDENCE FOR AN ELECTROGENIC PHOSPHATE PUMP

Uptake of phosphate from flowing solution and cell membraneelectrical potential differences (PD) have been followed simultaneouslyin the roots of Trifolium repens L. Intact plants were usedand it was found that uptake and PD were strongly influencedby the shoot. They were reduced by excision of the root, defoliationof the plant, and lowering the light intensity at the leaf surface.2, 4-Dinitrophenol caused the PD to decline by approximately40 mV. A close correlation between phosphate uptake and PD wasobtained over a wide range of conditions and removal of phosphatefrom the solution resulted in a decline in PD of about 35 mV.It was concluded that there is an electrogenic phosphate pumpin T. repens roots which is closely dependent on the carbohydratesupply from the shoot.     

Effect of sulphate deficiency on the plasma membrane polypeptide composition of Brassica napus

Highly purified oilseed rape (Brassica napus) root plasma membranefractions were prepared and their polypeptide patterns analysedby two-dimensional gel electrophoresis. Sulphur starvation enhancesthe sulphate uptake capacity of B. napus roots. The relativeabundance of several polypeptides increased significantly andspecifically after sulphur starvation. Several of them (37,38, 60, and 65 kDa), found in sulphur-starved plants, were moreabundant in a phase-partitioned membrane fraction treated withTriton X-100/KBr, indicating that they are intrinsic polypeptides.One polypeptide (47 kDa) was identified in the in vitro translationproducts of the roots mRNAs as specific for S-starved plants.It was also present among the intrinsic polypeptides specificfor —S plants. These plasma membrane polypeptides mightbe involved in sulphate uptake. Key words: Sulphate, sulphur-starvation, plasma membrane, polypeptides, root, transport     

Phosphorus uptake suppression in maize seedlings as related to humate supply

The effect of sodium humate on phosphorus uptake by maize seedlings devoid of the endosperm was studied. Short-term experiments have shown that a suppression of phosphate ion absorption occurs under the described conditions in the presence of humate without decreasing the weight of experimental plants. The effect reported is delayed and is enhanced by prolonging the preliminary phase of starvation. It varies with the pH of the medium and reduces under extreme conditions the phosphate release from the root to the medium.     

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (anitrate-based medium, not phosphate limited) were measured usingcells grown semi-continuously and in continuous culture. Netuptake of phosphate is proportional to external concentration.Growing cells at pH_o 10 have a net uptake rate of about 600pmol m^–2 s^–1 phosphate, but the isotopic flux for³²P phosphate was about 4 nmol m^–2 s^–1. There appearsto be a constitutive over-capacity for phosphate uptake. TheK_m and V_max, of the saturable component were not significantlydifferent at pH_o 7.5 and 10, hence the transport system probablyrecognizes both H₂PO^–₄and HPO^2–₄. The intracellularinorganic phosphate concentration is about 3 to 10 mol m^–3,but there is an intracellular polyphosphate store of about 400mol m^–3. Intracellular inorganic phosphate is 25 to 50kJ mol^–1 from electrochemical equilibrium in both thelight and dark and at pH_o 7.5 and 10. Phosphate uptake is veryslow in the dark ( 100 pmol m^–2 s^–1) and is light-activated(pH_o 7.51.3 nmol m^–2 s^–1, pH_o 10600 pmol m^–2s^–1). Uptake has an irreversible requirement for Mg²⁺in the medium. Uptake in the light is strongly Na⁺-dependent.Phosphate uptake was negatively electrogenic (net negative chargetaken up when transporting phosphate) at pH_o 7.5, but positivelyelectrogenic at pH_o 10. This seems to exclude a sodium motiveforce driven mechanism. An ATP-driven phosphate uptake mechanismneeds to have a stoichiometry of one phosphate taken up perATP (1 PO_{4 in}/ATP) to be thermodynamically possible under allthe conditions tested in the present study. (Received June 16, 1997; Accepted September 4, 1997)