Mixis strategies and resting eeg production of rotifers living in temporally-varying habitats

The organic phosphate pool of some Camargue sediments (South of France) was studied, after removal of inorganic phosphate, with Ca-NTA/dithionite (Fe bound phosphate) and Na-EDTA (Ca bound phosphate). The organic phosphate was divided into an acid soluble organic phosphate fraction (ASOP) and a residual organic phosphate fraction (ROP). The extraction of organic matter with 2.0 M NaOH (90 °C) from ROP yielded considerable quantities of Org-P. In this extract the presence of phytate (inositol hexa phosphate) could be demonstrated using phytase to hydrolyse the phytate. Phytate was shown to account for a considerable part of organic phosphate in sediments of freshwater marsh sediments as well as in the sediment of the brackish/salt water lake ‘Etang de Vaccares’. In laboratory experiments phytate was found to precipitate with all poly-valent cations tested. Furthermore, phytate was found to be strongly adsorbed onto Fe(OOH), which may explain its accumulation and its stability in sediments. Considerable quantities of ASOP were found; the chemical stucture of this pool remains unknown.     

The distribution of phosphate over iron-bound and calcium-bound phosphate in stratified sediments

Release of phosphate from, and adsorption ontosediments is calculated as a chemical equilibriumbetween dissolved o-phosphate and two solidphosphates, i.e. iron- and calcium-bound phosphate.Organic phosphates play a minor role, if any at all.Using chemical equilibrium equations, the distributionof the two solid inorganic phosphates is calculatedfrom the accumulated phosphate quantity as function oftime and depth in sediment layers of shallow lakes orwetlands. It is shown that this distribution dependson water depth, pH, Ca²⁺ concentration in thewater, Fe(OOH) concentration in the sediments andmaximal binding capacity of the sediments. Bycomparing values of dissolved phosphate at differentpH values, it is shown that acidification, whichusually takes place in hypolimnia, will cause releaseof phosphate, which is not necessarily dependent onthe redox potential. The release does depend on pH,Ca²⁺ concentration in the water, CaCO₃concentration in the sediments and the saturationstage of the two P-pools in the surface layers ofthese sediments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative determination of phytate in the presence of high inorganic phosphate

The review on the determination of phytate and inositol phosphates by Oberleas (1) indicates that most methods for the determination of phytate are derived from the method of Heubner and Stadler (2). This method is based on the principle that ferric ion forms a stable complex with phytate in dilute acid solution and is the only phosphate compound, at least in significant concentration in nature, with this property. However, the phytate values were high when we applied the procedure of Oberleas (3) to samples with high inorganic phosphate content such as rat feces or semipurified rat diets. This appeared to be a result of inorganic phosphate coprecipitating with ferric phytate.     

Available phosphorus in lake sediments in The Netherlands

The amount of phosphorus available to algae in the sediments of four lakes in the western part of the Netherlands has been assessed by means of chemical extraction and bioassay techniques. In addition to direct chemical sediment analyses, extractions were carried out with an NTA column method and a stepwise NH₄ Cl-NaOH-HCI shaking method, the latter supposedly separating the weakly bound, the Fe- and Al-bound and the Ca-bound phosphates in the sediments. Bioassays, with sediment as the sole source of P, were made withScenedesmus quadricauda in modified Skulberg's 28 medium to determine the amount of phosphates available to algae.The average total P concentration of the sediments varied from 0.8 to 3.6 mg P g^–1 dry wt and correlated well with the net external P loading of the lakes. Uptake of P by algae in the bioassays varied from 0.4 to 36% — while NTA extracted 36–69% of the total P. The ratio NH₄Cl extracted/ NaOH extracted/ HCI extracted phosphates is different from lake to lake, although in all lakes the highest extractions (27–62% of total P) are found in the NaOH fraction. However, in the peaty sediments of these lakes, the NaOH step extracted not only the Fe- and Al-bound phosphates but, also, large amounts of humus compounds. Hence, this fraction also contains non-available organic P.The results are related to soil type and chemical characteristics of the sediments, and compared with data from other authors. A positive correlation was found between phosphate available to algae and NTA- and NaOH-extractable P, but the correlation with total phosphorus was higher. Moreover, algal-extractable P proved to be positively correlated with total iron and clay content and negatively with the amount of organic matter.It is concluded that the sediments in the investigated lakes show great variability and that the chemical extraction techniques cannot replace the bioassays to assess the amount of phosphorus available to algae.     

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.     

Anaerobic Phosphate Uptake by Barley Plants

Considerable uptake of phosphate by both the shoot and roothas been demonstrated for young barley plants with their rootsin anoxic culture solution at concentrations of 1 to 10 µMorthophosphate. Consideration of the free space and passivetranspirational uptake indicates an accumulatory process, andthe immediate efflux caused by respiratory inhibitors supportsthis. Shoot uptake is much less at higher external concentrationsof phosphate and at o.I mM was only 14 per cent of the control.The root accumulation process was unimpaired at an externalconcentration of 1 µM phosphate when the whole plant wassubjected to anaerobic conditions (shoot illuminated) but undersimilar conditions at a concentration of 100 µM a considerableefflux of phosphate occurred. Analysis of the fate of phosphatetaken up from anoxic solution of phosphate (10 µM) indicatedthat there was a reduction in the level of inorganic phosphateafter 4.5 h and steady rise in sugar phosphates up to 6 h witha marked increase in the levels of glucose-6-phosphate, fructose-6-phosphate,and the phosphoglycerate fraction.     

Ca(2+)-inositol phosphate chelation mediates the substrate specificity of beta-propeller phytase

Inositol phosphates are recognized as having diverse and critical roles in biological systems. In this report, kinetic studies and TLC analysis indicate that beta-propeller phytase is a special class of inositol phosphatase that preferentially recognizes a bidentate (P-Ca(2+)-P) formed between Ca(2+) and two adjacent phosphate groups of its natural substrate phytate (InsP(6)). The specific recognition of a bidentate chelation enables the enzyme to sequentially hydrolyze one of the phosphate groups in a bidentate of Ca(2+)-InsP(6) to yield a myo-inositol trisphosphate (InsP(3)) and three phosphates as the final products. A comparative analysis of (1)H- and (13)C NMR spectroscopy with the aid of 2D NMR confirms that the chemical structure of the final product is myo-Ins(2,4,6)P(3). The catalytic properties of the enzyme suggest a potential model for how the enzyme specifically recognizes its substrate Ca(2+)-InsP(6) and produces myo-Ins(2,4,6)P(3) from Ca(2+)-InsP(6). These findings potentially provide evidence for a selective Ca(2+)-InsPs chelation between Ca(2+) and two adjacent phosphate groups of inositol phosphates.     

Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific, secretory expression in transgenic soybean plants

Phytases release inorganic phosphates from phytate in soil. A gene encoding phytase (AfPhyA) was isolated from Aspergillus ficuum and its ability to degrade phytase and release phosphate was demonstrated in Saccharomyces cerevisiae. A promoter from the Arabidopsis Pky10 gene and the carrot extensin signal peptide were used to drive the root-specific and secretory expression of the AfPhyA gene in soybean plants. The phytase activity and inorganic phosphate levels in transgenic soybean root secretions were 4.7 U/mg protein and 439 μM, respectively, compared to 0.8 U/mg protein and 120 μM, respectively, in control soybeans. Our results demonstrated the potential usefulness of the root-specific promoter for the exudation of recombinant phytases and offered a new perspective on the mobilization of phytate in soil to inorganic phosphates for plant uptake. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guilan Li and Shaohui Yang authors contribute equally to the paper.     

Quantitative trait loci analysis of phytate and phosphate concentrations in seeds and leaves of Brassica rapa

Phytate, being the major storage form of phosphorus in plants, is considered to be an anti-nutritional substance for human, because of its ability to complex essential micronutrients. In the present study, we describe the genetic analysis of phytate and phosphate concentrations in Brassica rapa using five segregating populations, involving eight parental accessions representing different cultivar groups. A total of 25 quantitative trait loci (QTL) affecting phytate and phosphate concentrations in seeds and leaves were detected, most of them located in linkage groups R01, R03, R06 and R07. Two QTL affecting seed phytate (SPHY), two QTL affecting seed phosphate (SPHO), one QTL affecting leaf phosphate and one major QTL affecting leaf phytate (LPHY) were detected in at least two populations. Co-localization of QTL suggested single or linked loci to be involved in the accumulation of phytate or phosphate in seeds or leaves. Some co-localizing QTL for SPHY and SPHO had parental alleles with effects in the same direction suggesting that they control the total phosphorus concentration. For other QTL, the allelic effect was opposite for phosphate and phytate, suggesting that these QTL are specific for the phytate pathway.     

Relation between algal available phosphate in the sediments of the River Garonne and chemically-determined phosphate fractions

Concentrations of total and inorganic phosphate were measured in forty-seven sediments of the River Garonne (France) using an anion resin, NaOH 0.1 M, Ca-NTA and Na-EDTA. Algal available P was measured using Scenedesmus crassus. The sediments presented a wide range of Tot-P concentrations (226 to 923 μg g⁻¹). Resin P varied between 1.1 to 55.5 μg g⁻¹ and NaOH-P between 3.5 to 262 μg g⁻¹. NTA-P and EDTA-P varied between 13.9 and 178 μg g⁻¹ and between 14.4 to 261 μg g⁻¹ respectively. Algal available P varied between 8.8 to 262 μg g⁻¹/ All forms of P are highly correlated and specially with algal available P (r = 0.70, 0.77, 0.88 and 0.77 for resin-P, NaOH-P, NTA-P and EDTA-P respectively). Nevertheless the partial correlation between EDTA-P and algal available P dropped to 0.05 when the concentration of NTA-P was considered as constant, indicating a spurious correlation with apatitic phosphate (extracted by Na-EDTA). Because of the strongest R ² and the lowest standard error of estimate, phosphate extracted by Ca-NTA can be considered as the best predictor of algal available phosphate.     

Sorption of Cadmium by Microorganisms in Competition with Other Soil Constituents

The fate of cadmium in soil is influenced to a great extent by microbial activity. Microorganisms were compared with abiotic soil components for their ability to sorb Cd from a liquid medium. When the same amount (on a dry weight basis) of bacterial cells (Serratia marcescens and Paracoccus sp.), clay (montmorillonite), or sand was separately incubated in 0.05 M phosphate buffer, pH 7.2, containing 10 ppm of Cd (10 μg/ml), bacterial cells removed the largest quantity of Cd. Dead cells sorbed much more Cd from the medium than live cells. A comparative study of Cd removal from the medium by seven soil bacteria and four fungi did not indicate appreciable differences. With increasing microbial biomass, the relative efficiency of 0.1 M NaOH as an extractant of sorbed Cd increased, whereas the extraction efficiency of 0.005 M DTPA (diethylenetriaminepentaacetic acid) decreased. It appeared that NaOH and DTPA extracted different chemical forms of Cd. This assumption was supported by vastly different correlation coefficients in the relative amount of Cd extracted by the two solvents.     

Phytate as the sole phosphate source for growth of Tetrahymena

Phytate, the storage form of phosphate in seeds and grains, is a major form of environmental phosphate loading from fertilizer inputs and agricultural runoff. We have investigated the ability of Tetrahymena populations to grow on phytate as their sole phosphate source. Populations grew equally well in chemically defined medium with phosphate and medium in which the phosphate was replaced with phytate in comparable concentrations between 0.5 mM and 6 mM. Intracellular phytate concentrations of cells grown in phytate showed a 4-6-fold increase over those grown in phosphate when measured during the late stage of exponential growth. These results demonstrate that phytate can provide a source of adequate phosphate for sustained growth in phytate-rich environments.     

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg⁻¹ protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg⁻¹ of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Observation of anoxic water mass in a tropical reservoir: the Cirata Reservoir in Java,Indonesia

We conducted a transect survey of water quality and bottom sediments in a large tropical reservoir, the Cirata Reservoir, located on the Citarum River, West Java, Indonesia. In the main basin of this reservoir, the surface water contained high concentrations of chlorophyll a, up to 48 μg l⁻¹, and most of the water body was occupied by thick anoxic water. The thickness of the surface oxygenated water was only 5–7 m, whereas that of the anoxic water mass was more than 70 m. The concentrations of phosphate and ammonia were quite high in the anoxic hypolimnion. The reasons for the formation of the huge anoxic water mass include the oligomictic status of circulation, a relatively weak mixing caused by topography, high hypolimnion temperature, and high loads of organic matter. The carbon/nitrogen (C/N) ratios and the carbon stable isotope ratios of sediments indicated that the major source of organic carbon in the sediments was algal production in the reservoir and fish culture activity. The mechanism of eutrophication in the reservoir is also discussed.     

Relation between phosphate and organic matter in fish-pond sediments of the Deroua fish farm (Béni-Mellal, Morocco): implications for pond management

An experimental approach of the phosphate exchange across the water–sediment interface in fish ponds of the Deroua farm (Béni-Mellal, Morocco) is needed to understand the phosphate dynamics in these ponds in relation to their water quality. During this study, we conducted experiments to determine the P-fractions of the different pond sediments and to estimate the release from these sediments of phosphate available for algal uptake. We also determined the amount of phosphate needed to saturate the sediments of two different fish ponds under anoxic and oxic conditions. Phosphate release from sediments comes mainly from Fe(OOH)P and is more important in ponds lined with sheets. The accumulation of organic matter in sediments favours the installation of anoxic conditions and enhances the phosphate release from CaCO₃P, labile in these pond sediments. Under experimental conditions, org-P plays a minor role in the P-release. Oxic conditions, to the contrary, have an inhibitory effect on the P-release from sediments. About 80–98% of the P-adsorbed onto different pond sediments was recovered in the inorg-P-fractions. Aeration induces the oxidation of FeS to Fe(OOH) which can adsorb phosphate from solution. Besides, the presence of bacteria in pond sediments was essential to promote phosphate release under anoxic conditions by controlling the oxidation state of iron and the mineralization of the organic matter. Sheet-lined ponds, when insufficiently dried, accumulate a large quantity of organic matter in their sediments. After a decrease in pH, P is released from CaCO₃P and enhances the phytoplankton productivity responsible for renewed accumulation of organic matter. Org-C concentrations in sediments over 20 mg g^–1 d.w. favour the formation of toxic factors (Fe²⁺, Mn²⁺, NO₂ ^– and H₂S) harmful for carp growth. An extended period of drying efficiently enhances the mineralization of organic matter.     

Molecular and physiological characterisation of a 3-phytase from soil bacterium<Emphasis Type="Italic"> Klebsiella</Emphasis> sp. ASR1

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis"

Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.     

A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase

Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Br?nsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.