Alkaline phosphatase activity in two planktonic desmid species and the possible role of an extracellular envelope

1. Cosmarium abbreviatum var. planctonicum , a desmid from oligo‐mesotrophic lakes, had a higher maximum alkaline phosphatase activity (APA) and affinity constant under both continuous and pulsed inorganic phosphorus (P_i) limitation than Staurastrum chaetoceras , a desmid predominantly encountered in eutrophic lakes.
2. APA of both species increased when measured in starved cells subjected to pulsed P_i conditions when compared to continuous P_i limitation.
3. The portion of extracellular relative to cellular APA was higher in S. chaetoceras than in C. abbreviatum , indicating that S. chaetoceras secreted the enzymes more readily into its environment.
4. The difference in APA could explain the dominance of C. abbreviatum during competition between these two species under conditions of continuous organic P (P_o) limitation, but not the outcome under a pulsed P_o shortage. The dominance of S. chaetoceras in the latter experiment can, however, be explained by species‐specific P_i‐uptake characteristics.
5. After a saturating pulse of P_i, no increase in P_i in the extracellular mucus layer of C. abbreviatum was found and it was therefore concluded that the mucilage sheath does not store P. However, the sheath could have a main function as an accumulation site of cellular APA, providing the cell with P_i.     

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (P_i) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca²⁺]. Cultured fetal rat cortical neurons constitutively import P_i, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular P_i is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular P_i dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. P_i similarly increased neuronal survival after exposure to kainic acid or H₂O₂. P_i-exposed neurons had higher basal intracellular [P_i], [ATP], and [GSH], and slightly lower cytosolic free [Ca²⁺], compared with P_i-deprived neurons. P_i-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H₂O₂, compared with P_i-deprived neurons. These findings demonstrate that changes in extracellular and intracellular P_i can affect neuronal survival after excitotoxic or oxidative insults.     

Effects of salinity and phosphate on ion distribution in lupin leaflets

Lupin ( Lupinus luteus L. cv. Weiko III) were grown in nutrient solution over a range of inorganic phosphate (P_i) concentrations, with or without 50 m M NaCl. Plants with high P_i (2 m M ) and salt showed progressive leaf necrosis and had higher concentrations of total phosphate than plants grown with high P_i alone. Most of the extra total phosphate in salt treated plants was in the P_i form. P_i supply did not influence Na⁺, K⁺ or Cl⁻ concentrations in epidermal vacuoles or mesophyll cells. However, epidermal vacuoles accumulated more monovalent cations (Na⁺ and K⁺) than Cl⁻, and in vacuoles of plants grown with 0.1 m M P_i additional P_i was accumulated, possibly to maintain charge balance. Plants grown with 2 m M P_i did not accumulate additional P_i in epidermal vacuoles, but showed higher phosphorus levels in cell walls. It is suggested that at moderate phosphorus concentrations P_i plays a role in epidermal osmotic adjustment, possibly explaining the beneficial role of additional phosphorus on salt stressed plants. At high P_i supply with salt, P_i does not contribute to osmotic adjustment and instead accumulates in cell walls. However, the cause of leaf damage under conditions of high phosphorus supply and salinity is still not entirely clear.     

Photosynthetic response of P-deficient Gracilaria tenuistipitata under two different phosphate treatments

Phosphorus-deficient Gracilaria tenuistipitata Zhang et Xia was cultured for 15 days at two different inorganic phosphate (P_i) concentrations: 3 μM (low P_i treatmenl) or 30 μM phosphate (high P_i treatment). The amount of ribulose-l,5-bisphosphate carboxy-lase/oxygenase (Rubisco), phycobiliproteins, Chl a and total soluble proteins were higher in the high P_i than in the low P_i treatment. The total N content of the low P_i plants was lower than in plants grown at high P_i concentrations whereas the amount of total C was highest in low P_i plants. The increase of Rubisco content in the high P_i treatment (3-fold) was parallel to the enhancement of the maximum photosynthetic rate which increased 5-fold. This correspondence was also found in the low P_i treatment in which Rubisco content and maximum photosynthesis did not change significantly from the initial values. The photosynthetic efficiency was also higher at high than at low P_i. The high P_i plants also showed higher dark respiration and growth rates. The data presented here suggest that marine macroalgae submitted to P_i deficiency exhibit a decrease in growth caused not only by P_i implication on energy transfer in photosynthesis and respiration, but also by the diminution of the amount of photosynthetic pigments and Rubisco.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

The relationship between phosphate status and cyanide-resistant respiration in bean roots

Bean ( Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of P_i deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased P_i content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in P_i-deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to P_i-deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in P_i-deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

Abstract We examined phosphate (P_i) uptake by two well-characterized microorganisms: a green alga ( Selenastrum capricornutum ) and a heterotrophic yeast ( Rhodotorula rubra ). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of P_i. We found that, under these conditions, both organisms had very high transport rates for P_i. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store P_i more efficiently than the alga under dilute P_i continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.     

The influence of salinity on phosphate uptake and distribution in lupin roots

Treeby, M. T. and van Steveninck, R. F. M. 1988. The influence of salinity on phosphate uptake and distribution in lupin roots. - Physiol. Plant. 72: 617–622.
The uptake and distribution of phosphate in lupin ( Lupinus luteus L. cv. Weiko III) roots under moderate salt (NaCl) stress was studied. Vacuolar inorganic phosphate (PJ concentrations in high phosphate plants were decreased by salt, although whole root P| was unaffected. In low phosphate plants, vacuolar P_i was unaffected by salt while whole root P_i was increased. Phosphate uptake was not altered by salt in high phosphate plants, but was depressed in low phosphate plants. These observations lead to the conclusion that in high phosphate plants P_i accumulates in cytoplasm and/or stele, ultimately giving rise to phosphate toxicity in shoots. Increasing phosphate supply had no effect on Na⁺ accumulation in root cell vacuoles in the epidermis or cortex, but the concentration of Cl⁻ in endodermal vacuoles was lowered.     

Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.)

We investigated (1) the effect of constant and altered inorganic phosphate (P_i) supply (1–100 mmol m^–3) on proteoid root production by white lupin ( Lupinus albus L.); and (2) the variation in citrate efflux, enzyme activity and phosphate uptake along the proteoid root axis in solution culture. Proteoid root formation was greatest at P_i solution concentrations of 1–10 mmol m^–3 and was suppressed at 25 mmol m^–3 P_i and higher. Except at 1 mmol m^–3 P_i, the formation of proteoid roots did not affect plant dry matter yields or shoot to root dry matter ratios, indicating that proteoid roots can form under conditions of adequate P supply and not at the expense of dry matter production. Plants with over 50% of the root system as proteoid roots had tissue P concentrations considered adequate for maximum growth, providing additional evidence that proteoid roots can form on P-sufficient plants. There was an inverse relationship between the P_i concentration in the youngest mature leaf and proteoid root formation. Citrate efflux and the activities of enzymes associated with citric acid synthesis (phosphoenolpyruvate carboxylase and malate dehydrogenase) varied along the proteoid root axis, being greatest in young proteoid rootlets of the 1–3 cm region from the root tip. Citrate release from the 0–1 and 5–9 cm regions of the proteoid root was only 7% (per unit root length) of that from the 1–3 cm segment. Electrical potential and ³²P_i uptake measurements showed that P_i uptake was more uniform along the proteoid root than citrate efflux.     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

PRODUCTION OF MUCILAGE BY THE ADRIATIC EPIPELIC DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE) UNDER NUTRIENT LIMITATION

The carbon partitioning of the epipelic diatom Cylindrotheca closterium (Ehrenberg) Reiman and Lewin isolated from the Adriatic Sea was studied in the laboratory under varying scenarios of nutrient limitation. Total number of cells, photosynthesis measured at 695 μmol photons·m ^{− 2}·s ^{− 1} irradiance (P_{695- μ mol}), chlorophyll ( a + c ) content, respiration, extracellular polymeric substances (EPS), total particulate carbohydrate (TPC), and dissolved carbohydrate were evaluated under nitrogen and phosphorus deficiencies in culture. The highest total number of cells was found in the control, whereas the nitrogen-limited treatment showed the lowest value. During the transition phase of growth, photosynthesis in the nitrogen-limited treatment was 3-fold lower than in the phosphorus-limited treatment and 4-fold lower than in the control. Differences in respiration rates and chlorophyll ( a + c ) content were even more marked. Dissolved carbohydrate remained the same in all the treatments, whereas during the transition and stationary phase, EPS presented the highest values under phosphorus limitation and the lowest in the control treatment. The production of EPS was closely linked to the periods of carbon assimilation (transition phase) in the nutrient depleted treatments, especially in the phosphorus-limited treatment. These results point out the relevance of the nutrient imbalance (nitrogen or phosphorus) in the production of EPS by the benthic or resuspended diatoms and suggest that these diatoms play an important role in nutrient-unbalanced systems like sediments or marine snow.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Exoprotease activity of Leuconostoc oenos in stress conditions

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X₂L isolated from wine. Starved cells after 2 h of incubation at 30 °C in citrate buffer, 0.05 mmol 1⁻¹ pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l⁻¹ SO₂ and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l⁻¹ Ca²⁺ and Mg²⁺ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3'-5'-phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.     

Competition for dietary carbohydrates between streptococci in dental plaque

Abstract The predominant microorganisms in dental plaque, i.e., streptococci and actinomycetes, are carbohydrate fermenters. In the natural environment these organisms experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. Recently, we have studied the competition between pairs of oral Streptococcus species in the chemostat under continuous glucose or sucrose limitation and under glucose pulsed conditions.
In the present study we have investigated the competition for dietary carbohydrates in dental plaque of gnotobiotic rats infected with the same combinations of streptococci. The rats were exposed to similar regimes of carbohydrate administration as in the chemostat by incorporation of low molecular carbohydrates in the diet and drinking water. Addition of glucose to the diet favoured the organism with the highest q _max for glucose. This result parallels the outcome of earlier competition experiments in glucose-pulsed chemostats, which showed that the organism with the highest q _max glucose became dominant in the cultures. Previous observations that bacteriocin production and extracellular glucan synthesis are major ecological factors for oral streptococci were also confirmed in this experiment.     

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P_i) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P_i transport and the activity of P_i transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P_i transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P_i transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P _i transporter downregulated ( ptd1 ) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P_i transporters in mycorrhizal roots.     

Depletion of stromal Pi induces high 'energy-dependent' antenna exciton quenching (qE) by decreasing proton conductivity at CFO-CF1 ATP synthase

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P_i), which results in down-regulation of light capture via the exciton quenching (q_E) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P_i depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF_O-CF₁ ATP synthase to protons ( g _H⁺) is decreased, retarding proton efflux from the lumen. Sequestering of P_i by mannose infiltration increased sensitivities of q_E and pmf to LEF. The effects were attributable to decreases in g _H⁺, but not to CEF1 and were largely reversed by subsequent P_i feeding. Rapid recovery of g _H⁺ in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson–Calvin cycle intermediates or lowering of stromal P_i below its K _Mat the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.