Unfolding and refolding of Aspergillus niger PhyB phytase: role of disulfide bridges

The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.     

Salt effect on the pH profile and kinetic parameters of microbial phytases

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.0. A high concentration of sodium chloride affected the activity of fungal phytase in the pH 3-4 range and shifted the pH optimum to 2.0 from 5.5 in Escherichia coli phytase. As expected, both of the microbial phytases were inhibited by sodium fluoride at acidic pH values. Because the Km for phytate increased nearly 2-fold for fungal phytase while Vmax increased about 75% in a high concentration of sodium chloride, it is possible that salt enhanced the product to dissociate from the active site due to an altered electrostatic environment. Modeling studies indicate that while the active site octapeptide's orientation is very similar, there are some differences in the arrangements of alpha-helices, beta-sheets, and coils that could account for the observed catalytic and salt effect differences.     

In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation

Activities of phytase, a pH 6.0 optimum nonspecific phosphomonoesterase and phosphodiesterase assayed toward bis(p-nitrophenyl)phosphate (phosphodiesterase I) and against p-nitrophenylphosphorylcholine (phosphodiesterase II), were partially purified from mycelial extracts of Aspergillus niger AbZ4 cultivated on a molasses medium by a liquid surface fermentation method. After elimination of phosphate from the medium, 7.3- and 3.5-fold enhancements in specific activities of phytase and phosphodiesterase II were observed. Efficacies of mycelial protein fractions in dephosphorylating a wheat-based broiler feed were determined in vitro according to a procedure that simulated digestion in the intestinal tract of poultry. The addition of 0.052 mg of protein from fractions, each of which was high in either pH 6.0 optimum phosphomonoesterase, phosphodiesterase I, phosphodiesterase II, or phytase per gram of a feed sample resulted in the enhancement of phosphorus release by 10, 11, 27, and 88%, respectively. In the presence of an excess of commercial phytase, the addition of the mycelial fraction high in phytase increased the dephosphorylation rate by 56%. The fraction high in phosphodiesterase II enhanced feed dephosphorylation by 8% in the presence of an excess of commercial phytase and commercial acid phosphatase.     

低分子量有机酸对红壤磷酸单酯酶活性的影响

采用室内模拟试验研究了低分子量有机酸(柠檬酸、草酸、苹果酸、酒石酸)对红壤磷酸单酯酶活性的影响。结果表明,低浓度有机酸(<1μmol g-1)对磷酸单酯酶活性有促进作用,且促进作用大小依次为柠檬酸≈草酸>苹果酸>酒石酸;而高浓度有机酸(>5μmol g-1)则为抑制作用,且抑制作用大小依次为柠檬酸>草酸>苹果酸>酒石酸。当体系pH趋向酶促反应最佳pH时,磷酸单酯酶活性增强;反之,当体系pH远离酶促反应最佳pH时,磷酸单酯酶活性减弱。有机酸根一方面通过羧基的辅助作用提高磷酸单酯酶活性;另一方面通过释放土壤A l3+、Fe3+等金属离子,对土壤磷酸单酯酶活性有一定的抑制作用。     

Effect of arsenic contamination on bacterial and fungal biomass and enzyme activities in tropical arsenic-contaminated soils

The importance of assessing the impacts of soil arsenic (As) contamination on microbial properties lay on the fact that microbes are instrumental in nutrient cycling and are therefore indicators of soil quality. In this study, soil chemical extraction methods were used to extract labile and freely exchangeable As (water-soluble As and sodium bicarbonate-extractable As), amorphous/crystalline Fe and Mn oxide-bound As (acid ammonium oxalate-extractable As and hydroxylamine hydrochloride-extractable As), and their impacts on microbial biomass (microbial biomass C, total bacterial and fungal biomass, active bacterial and fungal biomass), enzyme activities representing four major soil biogeochemical cycles, i.e., C (β-glucosidase activity), N (urease activity), P (acid phosphomonoesterase activity), S (acryl-sulfatase activity), and microbial activity (fluorescein diacetate hydrolysis and dehydrogenase activity) were investigated in As-contaminated soils of Ambagarh Chauki block, Chhattisgarh, Central India. The results revealed that the majority of the As in soils resided in the Fe/Mn oxide-bound fraction. The microbial biomass C, total and active fungal biomass, and enzyme activities were significantly inhibited by all the forms of As. However, water-soluble As, even though occupying only a small portion of the total As (0.9–2.9 %), exerted the greatest impact. Interestingly, total and active bacterial biomass was not significantly affected by As toxicity, suggesting their resistance to As. Urease activity was not affected by As pollution.     

Behaviour of plant‐derived extracellular phytase upon addition to soil

The behaviour of phytase after addition to three soil types with different sorption capacities was investigated. Phytase was collected from the roots of transgenic Arabidopsis thaliana that express a phytase gene from Aspergillus niger. Phytase activity in solution and on the solid phase of the soil was monitored over time. Phytase added to the solution phase of a soil suspension (1:20, w/v) was almost completely lost within 10 min in all soil types, while phytase in non-soil controls remained active in solution. Phytase activity lost from solution was recovered on the soil solid phase, suggesting rapid adsorption of the enzyme. Adsorption of phytase was less in soil taken from the rhizosphere of transgenic plants expressing phyA, indicating that the rhizosphere environment may help maintain phytase activity in solution. The activity of adsorbed phytase declined with time at a rate 2-4 times slower than that in the absence of soil. Adsorption of phytase in soils was highest at pH 4.5, which is below the reported isoelectric point (pI) of the Aspergillus phytase. As soil pH increased, adsorption decreased until, at pH 7.5, all phytase was in solution. Where phytase remained in solution, activity was maintained for at least 8 d. In contrast, the activity of adsorbed phytase was increasingly inhibited with time, particularly at low pH. By increasing the pH in soil suspensions, phytase that had remained active on the soil solid phase for 28 d was almost totally desorbed. Rapid immobilisation of phytase in soil may limit its capacity to interact with phytate, and this may compromise the ability of transgenic plants which exude phytase from their roots to acquire P from endogenous soil phytate.     

Phosphorus limitation in microorganisms leads to high phosphomonoesterase activity in acid forest soils

We addressed the mechanistic basis for a negative correlation between soil pH and phosphomonoesterase activity, often found in various soils. Also in the present study, a significant negative correlation was observed between soil pH and phosphomonoesterase activity measured at pH 6.5 in Japanese acidic forest soils (3 Inceptisols, 3 non-allophanic Andisols, and 2 allophanic Andisols). A hypothesis that higher activity of phosphomonoesterase in acid forest soils results from increased synthesis of phosphomonoesterase by microorganisms in response to P limitation was tested. Soils with lower pH showed a lower optimum pH for phosphomonoesterase activity and greater activity at the optimum pH than other soils. To assess nutrient limitations of the soil microbial community, the effects of addition of C, N, or P on phosphomonoesterase and dehydrogenase activities, which is an intracellular enzyme and the activity of which reflects overall microbial activity, were examined in the soil samples. Addition of P increased dehydrogenase activity in some forest soils. Also, microorganisms in some soils were co-limited by C, N, and P. Response ratios (RR) of phosphomonoesterase and dehydrogenase activities in P-amended soil to their activity in non-amended soil were used to evaluate the response of soil microorganisms to P limitation. The ratio of RR-dehydrogenase to RR-phosphomonoesterase was strongly correlated with phosphomonoesterase activity at the optimum pH (P < 0.01). The results indicate that P limitation accounts for higher phosphomonoesterase activity in the more acid forest soils.     

Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The Km for phosphotyrosine ranged from 465 to 590 microM as opposed to 135 to 160 microM for phytate. The Vmax, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 x 10(6) to 1.6 x 10(7) M(-1) s(-1)); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5 degrees C higher to 70 degrees C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.     

Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates

The effect of the soil environment on the mobility, stability and catalytic activity of phytase from two sources was compared, as these factors have important implications for the efficacy of enzyme function in soil. Phytase from an ascomycete fungus (Aspergillus niger) and a basidiomycete fungus (Peniophora lycii) was added to soil suspensions from three contrasting soils and activities in the solution and solid phase were monitored. The two enzymes were compared because the P. lycii phytase was known to have greater specific activity and a more acidic isoelectric point (pI) than A. niger and therefore predicted to have different adsorption characteristics. When added to soil suspensions buffered at pH 7.5, both phytases remained in solution in all of the soils. In contrast at near natural soil pH (pH 5.5), only the P. lycii phytase remained in solution, while the A. niger phytase was rapidly adsorbed to the soil solid phase. The extent of this adsorption was reduced, however, in a soil-dependent manner by prior addition of bovine serum albumin (BSA) to the soil suspensions. At the natural pH of the soil, the stability of the P. lycii phytase in soil solution was improved under sterile conditions, whereas degradation of the A. niger phytase was unaffected. Subsequently, P. lycii phytase was shown to be more effective at hydrolysing myo-inositol hexakisphosphate added to the soil. Moreover, the P. lycii phytase also hydrolysed more organic phosphate that was endogenous to a range of soils. This research indicates that the physicochemical properties of fungal phytases affect their mobility and temporal stability and their capacity to hydrolyse inositol phosphates in soil environments.     

Investigating the mechanisms for the opposing pH relationships of fungal and bacterial growth in soil

Soil pH is one of the most influential variables in soil, and is a powerful factor in influencing the size, activity and community structure of the soil microbial community. It was previously shown in a century old artificial pH gradient in an arable soil (pH 4.0-8.3) that bacterial growth is positively related to pH, while fungal growth increases with decreasing pH. In an attempt to elucidate some of the mechanisms for this, plant material that especially promotes fungal growth (straw) or bacterial growth (alfalfa) was added to soil samples of the pH gradient in 5-day laboratory incubation experiments. Also, bacterial growth was specifically inhibited by applying a selective bacterial growth inhibitor (bronopol) along the entire pH gradient to investigate if competitive interaction caused the shift in the decomposer community along the gradient. Straw benefited fungal growth relatively more than bacterial, and vice versa for alfalfa. The general pattern of a shift in fungal:bacterial growth with pH was, however, unaffected by substrate additions, indicating that lack of a suitable substrate was not the cause of the pH effect on the microbial community. In response to the bacterial growth inhibition by bronopol, there was stimulation of fungal growth up to pH 7, but not beyond, both for alfalfa and straw addition. However, the accumulation of ergosterol (an indicator of fungal biomass) during the incubation period after adding alfalfa increased at all pHs, indicating that fungal growth had been high at some time during the 5-day incubation following joint addition of alfalfa and bronopol. This was corroborated in a time-series experiment. In conclusion, the low fungal growth at high pH in an arable soil was caused to a large extent by bacterial competition, and not substrate limitation.     

Phosphomonoesterase,phosphodiesterase, phosphotriesterase,and inorganic pyrophosphatase activities in forest soils in an alpine area: effect of pH on enzyme activity and extractability

We investigated different types of phosphatase activity (phosphomono-, phosphodi-, phosphotriesterase, inorganic pyrophosphatase) in five forest soils in Vorarlberg, Austria. Phosphatase activity was determined both in soils and in soil extracts prepared with different solutions (distilled water, 0.1M sodium pyrophosphate at pH 7, 0.1M sodium phosphate buffer/1M KCl at pH 6.5, and a modified universal buffer at pH 4, 6.5, 9, and 11). High phosphomonoesterase activity in these soils indicated a severe deficiency in available P. Acidic phosphomonoesterase prevailed over alkaline phosphomonoesterase activity. Phosphodiesterase was highest in the least acidic soil but no general trend towards an optimum pH was recognized. Phosphotriesterase activity was observed in only two of the five soils and favoured an alkaline optimum pH; this activity was not detected in strongly acid soils. Inorganic pyrophosphatase activity was high in soils with no phosphotriesterase. Phosphomonoesterase, phosphodiesterase and inorganic pyrophosphatase activities were much lower in soil extracts than in soils.     

Relative efficiency of fungal intra‐ and extracellular phosphatases and phytase

A comparison of intra‐ and extracellular acid phosphatase, alkaline phosphatase and phytase activity in six fungi is reported. A strong linear relationship between intra versus extracellular fungal acid phosphatase (R² = 0.94), alkaline phosphatase (R² = 0.96), and phytase (R² = 0.97) is observed. Three‐fourth of acid phosphatase were generally present inside the fungal cells and only 25 % were released extracellularly after a three weeks period. Phytase shows the reverse trend where thirty nine times higher extracellular phytase activity was noticed than present inside the fungal cells. The extracellular enzymes are found 60 % more efficient in the hydrolysis of phytin than their intracellular counterpart but they are at par in the hydrolysis of glycerophosphate. The results clearly demonstrated that phytase types of phosphatases mostly occur outside the fungal cells whereas most of the acid phosphatase and alkaline phosphatase are located inside the cells.<?show $6#>     

Phosphomonoesterase production and persistence and composition of bacterial communities during plant material decomposition in soils with different pH values

The aim of this work was to study the synthesis and persistence of acid and alkaline phosphomonoesterases in three soils with different pH values amended with ryegrass residues. The organic input increased soil respiration, as estimated by CO₂-C evolution in all soils. The ATP content of the three soils showed a 3-7-fold increase between 7 and 10 d in the different soils since the amendment. The dsDNA content of the three amended soils also peaked between 7 and 10 d, increasing by 2.5-3.5 times in the different soils. The bacterial species richness increased in the amended as compared to the control soils during the early stages (7-10 d) of organic matter decomposition, as indicated by the decreasing values of the Sørensen similarity index between the treatments in this period. Soil amendment increased the alkaline phosphomonoesterase activity by 6, 8 and 15 times in the Vallombrosa acidic, Romola neutral and Vicarello alkaline soil, respectively, whereas the acid phosphomonoesterase activity showed a 6-, 2- and 10-fold increase in the Vallobrosa acidic, Romola neutral and Vicarello alkaline soil, respectively. Phosphatase activities peaked between 4 and 10 d, depending on the activity and the soil considered, but activity of alkaline phosphomonoesterase was higher in alkaline soils and persisted longer than the acid phosphomonoesterase activities; the opposite occurred in the acid soil. During a 180 d decomposition period, both acid and alkaline phosphomonoesterase activities were related to dsDNA and ATP contents in all soils. Peaks of phosphmonoesterase activity coincided with the changes in the composition of the bacterial microflora, as detected by 16S-rDNA analysis, although no relationship between bacterial community composition and persistence of the phosphomonoesterase activities could be shown. It was concluded that acid and alkaline phosphomonoesterases are produced in greater amounts during plant residue decomposition, and that in acid soils acid phosphomonoesterase activity predominates and in neutral and alkaline soils alkaline phosphomonoesterase activity predominates. However, the persistence of newly produced enzymes is determinated by other factors such as soil texture, organic matter content and formation of soil colloid-enzyme complexes.     

The combined effects of cover crops and symbiotic microbes on phosphatase gene and organic phosphorus hydrolysis in subtropical orchard soils

P deficiency is a major obstacle for crop production in subtropical red soils in South China, and the hydrolysis of organic P (Po) is of great significance in these soils due to the immobilization of P by Fe and Al. Cover cropping in orchards and symbiotic microbial inoculation are considered to improve soil quality, including P status, however, their effects on the hydrolysis of Po is little known. In this study, five soil managements were established in a guava orchard in South China for two and a half years, including clean culture (CC), cover cropping with Paspalum natatu (PN), PN with arbuscular mycorrhizal fungal inoculation (PNA), cover cropping with Stylosanthes guianensis (SG), SG with rhizobial inoculation (SGR). Soil chemical, biochemical and microbial properties were analyzed. Results indicate that soil pH and SOM content tended to increase following cover cropping alone or with microbial inoculation. Po content was significantly elevated in PNA. Po fractionation revealed that cover cropping alone or with microbial inoculation significantly affected the contents of moderately labile Po (MLPo) and moderately resistant Po (FAPo). Enzyme assay indicated that cover cropping with microbial inoculation increased the activities of acidic phosphomonoesterase (ACP), neutral phosphomonoesterase (NP) and alkaline phosphomonoesterase (ALP), with ALP the most sensitive, although ACP activity dominated in red soils. Correlation analysis suggested a significantly positive relationship between ALP activity and MLPo or FAPo. PCR-DGGE profile of the alp-harboring bacterial community showed that cover cropping with S. guianensis and mycorrhizal inoculation to P. natatu promoted the bacterial diversity and/or species richness. For almost all the measured parameters, PN and SG were comparable, however, PNA was superior to SGR, indicating the stronger additive effect of arbuscular mycorrhizal fungus than that of rhizobia. Cat-PCA indicated that MLPo was the most influential factor on phosphomonoesterase. In general, this study suggests that, in subtropical orchards with red soil, cover cropping with microbial inoculation can improve the Po hydrolysis via the promoted alp-harboring bacterial community and then ALP activity. Our results also suggest that the combination of P. natatu and arbuscular mycorrhizal fungus is better than S. guianensis and rhizobia, which possesses practical significance for sustainable production in these orchards.     

Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes

The activity of wheat and Aspergillus niger phytases was determined following preincubation for 60 min at 37 degrees C alone or in the presence of pepsin or pancreatin to examine their ability to survive in the gastrointestinal tract. At pH 3.5 both phytases were stable, but at pH 2.5 wheat phytase rapidly lost activity. Following preincubation at pH 3.5 in the presence of 5 mg of pepsin/mL, A. niger phytase retained 95% of its original activity, whereas only 70% of the wheat phytase activity was recovered. The stability of A. niger phytase in the presence of pepsin was the same at pH 2.5 as at pH 3.5. Results similar to those with pepsin at pH 3.5 were obtained following preincubation of the phytases in the presence of pancreatin at pH 6.0.     

Soil pH and phosphatase activity

Abstract

Approximately twenty years before this study, a site that consisted of a mixed oak forest was harvested, cleared, and divided into three treatment areas consisting of approximately 20 acres each. The three areas were planted to oak (forest), grass (grassland) and corn (agricultural) respectively. The influence of pH on the rate of phosphatase activity was determined over a broad range of soil pH in soil sampled from each treatment area. Phosphomonoesterase activities were measured at a pH of 2 through 12 and phosphodiesterase activities determined at a pH of 4 through 12. In the forest soil only a acid phosphomonoesterase was detected whose pH optima was maximal at the measured soil pH of 4.9. A neutral phosphomonoesterase was found in the grassland soil, pH 6.6, with a broad pH optima ranging from 4.6 to 7.0, while the detection of an acid phosphatase and a alkaline phosphatase, with a pH optima of 4.8 and 11.0 respectively, was found associated with the agriculture soil with a measured pH of 7.2. Phosphodiesterase activity was optimum or near optimum at the measured pH of each soil examined. The released phosphatases apparently have different pH optima in relation to maximal activity suggesting the presence of different kinds of phosphomonoesterases and phosphodiesterases and perhaps that the enzymatic reaction in soil is catalyzed by more than one enzyme or by multiple forms of the same enzyme. The results of the study would indicate that a relationship exists between soil pH and (1) the synthesis and release of phosphatases in soil, (2) the complexion of the organisms producing the enzymes and (3) phosphatase stability or conformation. Based upon the results of the study, the analysis of phosphatase activity at the measured soil pH would seem to be a necessary part of any investigation designed to determine the contribution of phosphatase enzymes to the cycling of P.     

Water Treatment Residuals and Biosolids Co‐applications Affect Phosphatases in a Semi‐arid Rangeland Soil

Co‐application of biosolids and water treatment residuals (WTR) land has not been extensively studied but may be beneficial by sorbing excess biosolid‐borne or soil phosphorus (P) onto WTR, reducing the likelihood of off‐site movement. Reduction of excess soil P may affect the role of specific P‐cleaving enzymes. The research objective was to understand the long‐term effects of single co‐applications and the short‐term impacts of repeated co‐applications on soil acid phosphomonoesterase, phosphodiesterase, pyrophosphatase, and phytase enzyme activities. Test plots were 7.5 × 15 m with treatments consisting of three different WTR rates with a single biosolids rate (5, 10, and 21 Mg WTR ha^?1; 10 Mg biosolids ha^?1) surface co‐applied once in 1991 or reapplied in 2002. Control plots consisted of those that received no WTR–biosolids co‐applications and plots that received only 10 Mg biosolids ha^?1. Plots were sampled to a 5‐cm depth in 2003 and 2004, and soil phosphatases and phytase enzyme activities were measured. Soil phosphodiesterase activity decreased in WTR‐amended plots, and pyrophosphatase activity decreased with increasing WTR application rates. In contrast, acid phosphatase and phytase activity increased with WTR addition, with WTR application possibly triggering a deficiency response causing microorganisms or plants to secrete these enzymes. Biosolids and WTR co‐applications may affect enzymatic strategies for P mineralization in this study site. Reductions in phosphodiesterase activity suggest less P mineralization from biomass sources, including nucleic acids and phospholipids. Increased acid phosphatase and phytase activities indicate that ester‐P and inositol‐P may be important plant‐available P sources in soils amended with WTR.     

Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The phytase was successfully separated from the major acid phosphatase to an electrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (K _m = 38 μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45°C, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.     

Importance of plasmalemma ATPase in the retention and exclusion of inorganic ions

The experiments were focused on the question whether the plasmalemma ATPase activity (proton pump) has an influence on the efflux of major inorganic ion species. Efflux from roots of intact Trifolium pratense, Hordeum vulgare, Glycine max, and Zea mays was examined into a solution containing 100 μM CaCl₂ and 500 μM NH₄⁺ as sulfate in the control solution and 100 μM CaCl₂ and 500 μM NH₄⁺ as vanadate in the test solution. Vanadate being an inhibitor of the plasmalemma ATPase depressed significantly the H⁺ secretion of roots into the outer solution but had no major impact on the efflux of cation species. In the presence of vanadate significantly higher amounts of sulfate, phosphate, and nitrate were released into the outer solution by roots of soya and maize as compared with the control treatment (no vanadate). In the absence of vanadate, virtually no nitrate was released by all species examined whereas in the vanadate treatment significant amounts of NO₃^? were released. Vanadate inhibited the uptake of Cl^? in barley and maize and increased the uptake of Ca²⁺ in soya. It is concluded that the plasmalemma ATPase activity plays a major rule in the “ionic stat” of cells in providing protons to the apoplast for the reabsorption of sulfate, phosphate, and particularly nitrate which have leaked out of the cytosol.     

Surface-bound phosphatase activity in ectomycorrhizal fungi: a comparative study between a colorimetric and a microscope-based method

For the quantification of surface-bound phosphomonoesterase activity (SBPA) of fungi, roots, or mycorrhiza, a colorimetric method based on p-nitrophenyl phosphate (pNPP) is widely used. Unfortunately, this method does not reveal information about the localization of the surface-bound phosphomonoesterase (SBP). We introduce a method that localizes and quantifies SBPA in living hyphae of ectomycorrhizal fungi using confocal laser scanning microscopy of the hydrophilic substrate enzyme-labelled fluorescence (ELF-97) and compare it to the pNPP assay. ELF-97 turns into a strongly fluorescent precipitate upon activation by SBPA and forms bright fluorescent centres on the outer cell wall of the hyphae. Our data show that the enzymatic reaction is not substrate-limited during an incubation period of 15 min in fungal hyphae of Pisolithus tinctorius, Cenococcum geophilum, and Paxillus involutus. Image-processing routines determined the total intensity and the average number of the fluorescent ELF-97 centres per micrometre fungal hyphae of C. geophilum and Paxillus involutus. ELF-97 and pNPP detected similar variations of the SBPA at different pH values (3–7) during the measurement and different phosphorus (P) concentrations during the growth period of the fungi. The ELF-97 method revealed that C. geophilum and Paxillus involutus adapt in different ways to the variation of the P concentrations during the growth period by varying the number, the activity, or both properties of the SBP centres. The phosphatases show peak activities at different pH values, so the response of the fungal mycelium to varying P concentrations in soils is pH selective. In conclusion, ELF-97 is a promising substrate to reveal SBPA and adaptation strategies on a structural–physiological level.