HOT WATER EXTRACTABLE PHOSPHORUS—AN INDICATOR OF NUTRITIONAL STATUS OF PERIDINIUM CINCTUM (DINOPHYCEAE) FROM LAKE KINNERET (ISRAEL)?1

The intracellular levels of hot water extractable and total phosphorus were determined in the dinoflagellate Peridinium cinctum. f. westii (Lemm.) Lef. for natural samples from the bloom in Lake Kinneret and from laboratory cultures. Amounts of phosphorus (P) in the hot water fraction, relative to total cellular phosphorus, were similar in lake Peridinium and in cells grown in high ambient orthophosphate (P_i) media (3–6 mg P · l^?1). The absolute amounts of hot water extractable P in natural cell and those cultured at lower P_i concentrations (0.02–0.05 mg P · 1^?1) were similar, although average P_i in lake water were 4 μg · l^?1. Under most growth conditions the hot water extract contained approximately equal amounts of molybdate reactive phosphorus (MRP) and non-MRP. Short chain (6–9 units) polyphosphates (mol wt 630–950) probably constituted the bulk of the non-MRP pool, which was hydrolysable by alkaline phosphatase and may serve as a precursor for a more permanent P store. Intracellular P levels and distribution were not directly dependent on external P_i concentrations but may be determined by the N:P atomic ratio or overall external ionic milieu. Peridinium grown in low ambient P_i released significant amounts of non-MRP compounds. In Lake Kinneret, for at least most of the bloom period, Peridinium does not appear to be limited by P supply.     

Phosphatase production byAspergillus ficuum

Summary Effects of nutritional and cultural conditions on cell growth and phosphatase production byAspergillus ficuum were studied.A. ficuum produced high levels of phosphatases when grown on a basal medium that contained a minimal amount (2 mg/100 ml) of phosphorus in an acidic growth medium. The organism produced a nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The enzyme hydrolyzed a variety of phosphates and produced orthophosphate. The rate of phosphate hydrolysis was dependent on the pH of the reaction, where the pH optimum for acid phosphatase was 2.5 and that for phytase was 5.0. The organism slowly released the phosphatase, and the enzyme activity in the growth medium increased continually during a one-month growth period. For a high level of phosphatase production, low levels (1–5 mg%) of initial phosphorus were necessary and polyphosphates were the desired form rather than the monophosphate. The addition of surfactants, such as polyoxyethylene ethers and sodium oleate, to fungal culture medium markedly increased the level of phosphatase production.     

ACCUMULATION OF WATER SOLUBLE PHOSPHORUS AND HYDROLYSIS OF POLYPHOSPHATES BY CLADOPHORA GLOMERATA (CHLOROPHYCEAE)1,2

Concentrations of hot-water extractable phosphorus from most samples of Cladophora glomerata (L.) Kütz. were relatively high (0.06–0.68%) and correlated closely with total dissolved P in ambient Lake Michigan water. Cladophora was able to hydrolyze polyphosphates by enzymes found in intracellular, extracellular and cell wall fractions. The intracellular phosphatase activity is pH dependent with the optimal hydrolysis rate at pH 7.8. Secretion of phosphatases is affected by pH, with maximum rate at 7, but affected little by light intensity. Magnesium is the most effective metallic cofactor required for maximal rates of intracellular phosphatase activities.     

Response to Phosphate Deprivation in Brassica nigra Suspension Cells : Enhancement of Intracellular, Cell Surface, and Secreted Phosphatase Activities Compared to Increases in Pi-Absorption Rate

Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the activities of the other phosphatases. The cell surface acid phosphatase had a K_m of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions.     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Origin and production of phosphatases in the acid Lake Gårdsjön

The activity of acid phosphatases was followed for one year in Lake Gårdsjön as well as in the inlet and the outlet of the lake. A budget of the phosphatases was calculated, including an estimation of the production of phosphatases. The phosphatase activity was also measured in two basins upstream of L. Gårdsjön: the north basin and the south basin of L. Stora Hästevatten.The acid phosphatase activity was very high compared with reported alkaline phosphatase activities in other lakes. About 95% of the phosphatases in L. Gårdsjön was produced in the lake, and the production was highest in early summer.Small Chrysophyceae (< 10 µm) probably produced the majority of the acid phosphatases in the investigated lakes, and accordingly could be favoured in environments with low phosphorus supply due to their ability to produce large amounts of phosphatases.     

Are the banks a source of recolonization after disturbance: an experiment on aquatic vegetation in a former channel of the Rhône River

Hydrolysis of natural dissolved organic phosphorus (DOP) in three hardwater lakes of different trophic level was calculated from kinetic data of phosphatase activity (PA) in different size fractions. DOP as well as kinetics of PA were determined every fortnight in depth profiles during the year 1990. 60% of DOP was assumed to be suitable substrate for phosphatases. The rate of hydrolysis increased markedly with higher trophic level. Average hydrolysis rate of DOP in polytrophic lake Thaler See was 3.26 nM P min^–1 (6 µg P-PO₄ l^–1 h^–1). In oligotrophic Lake Herrensee, dissolved phosphatases were responsible for more than half of the total hydrolysis. In the other two lakes, bacterial and algal surface PA dominated hydrolysis in changing parts depending on kinetics and DOP concentration. The regeneration rate of phosphate by PA was compared to phosphorus (P) excretion rate of zooplankton. Excretion was calculated from zooplankton data and excretion equations from the literature. In oligotrophic Lake Herrensee, excretion by zooplankton recycled in average 18% of the phosphate amount which was hydrolysed from DOP by PA. With higher trophic level, relevance of P excretion from zooplankton decreased drastically.     

Seasonal and interannual dynamics of polyphenols in Myriophyllum verticillatum and their allelopathic activity on Anabaena variabilis

In 4 successive years, we investigated the seasonal and interannual variability of the total polyphenolic pool and of the individual polyphenolic compounds in Myriophyllum verticillatum, as well as their allelopathic activity in a small eutrophic lake. We tested whether nutrient availability explained interannual and seasonal changes in the production of polyphenols. There were no strong interannual variations in plant tissue carbon, nitrogen and phosphorus concentrations, while total phenolic compounds (TPC) significantly differed between years, especially in apical meristems (range: 38–122 mg g⁻¹  dry weight (DW)). Seasonal patterns, with maxima between May and July, changed between years. Partially confirming the carbon-nutrient balance hypothesis sensu Bryant et al. [Bryant, J.P., Chapin III, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357–368], we found correlations between TPC and the C/N (carbon/nitrogen) ratio in some but not all years, especially in apical meristems. Plant tissue phosphorus content accounted also for the variability in TPC in some years. Crude extracts of apical meristems always inhibited the growth of Anabaena variabilis, used as a target cyanobacterium. Plant TPC concentration and allelopathic activity were significantly correlated in all years except in 2005. Bioassay-directed fractionation of M. verticillatum extracts coupled with LC–MS analyses of the respective fractions revealed several isomers of HHDP-di- and -tri-galloylglucose apparently responsible for the allelopathic effects. The individual active compounds revealed a more distinct seasonal pattern compared to the pool of phenolic compounds in M. verticillatum, with a clear maximum in May, the ecologically most relevant period for inhibitory effects of submerged macrophytes on phytoplankton.     

SUPEROXIDE DISMUTASE ACTIVITY IN PERIDINIUM GATUNENSE IN LAKE KINNERET: EFFECT OF LIGHT REGIME AND CARBON DIOXIDE CONCENTRATION1

The activity of superoxide dismutase (EC 1.15.1.1, superoxide: superoxide oxidoreductase) (SOD) was determined in Peridinium gatunense Lemm. under natural and controlled conditions. SOD activity increased toward the end of the spring algal bloom in Lake Kinneret simultaneously with maximal photosynthetic activity and conditions of elevated ambient stress such as high irradiance. Activity staining of native polyacrylamide gel electrophoresis gels of bloom samples showed a similar pattern to the spectrophotometrically measured SOD. Both Mn SOD and CuZn SOD were present, however no Fe SOD was found in Peridinium. One of three isoenzymes of Mn SOD showed marked differential regulation of activity under stress. An increase in the quantity of the 32-kDa Mn SOD polypeptide during the bloom was found to be unrelated to senescence; it was assumed that this polypeptide was induced by stress. Thus, SOD in Peridinium undergoes physiological and molecular acclimation to seasonal environmental changes. When Peridinium was exposed to various O₂ and CO₂ concentrations in culture, CuZn SOD significantly increased under high C0₂ concentrations and normoxic conditions (20% O₂). However, at high irradiances, Peridinium cultures exposed to low and high CO₂ concentrations also had similar CuZn SOD activity. It was concluded that stressful irradiance is the overriding cause of increased SOD activity in both lake samples and in cultures of Peridinium.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na₂HPO₄ at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl₂ at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Predicting concentration of total phosphorus and chlorophyll a in a lake with short hydraulic residence time

The relationship between total phosphorus and chlorophyll a concentration was determined for Skinner Lake, Indiana over an annual cycle in 1978–79. Total nitrogen:total phosphorus ratios in the epilimnion ranged from 19 to 220 suggesting a phosphorus-dependent algal yield in the epilimnion. Approximately 90% of annual TP loading reached the lake via streamflow, and 93% of this entered during snowmelt and spring-overturn periods. At that time incoming water flushed the lake 2.4 times. Atmospheric loading accounted for 1.4% of annual TP load. Internal hypolimnetic TP loading occurred during summer stratification. Mean [chl a] for the ice-free period was 15.15 mg m^–3, within the range expected for eutrophic lakes.The 1978–79 data were used in conjuction with the Vollenweider & Kerekes (1980) model to produce a model specific for the Skinner Lake system. The model predicted mean epilimnetic total phosphorus and chlorophyll a concentrations from mean total phosphorus concentration in inlet streams and from lake water residence time during the period of spring overturn and summer stratification. The Skinner-specific model was tested in 1982 and it closely predicted observed mean epilimnetic [TP] and [chl a] during the ice-free period. This study shows that variability in lake models which average data over an annual period can be reduced by considering lake-specific seasonal variation in hydrology and external TP loading.     

Response of root and sediment phosphatase activity to increased nutrients and salinity

Wetlands of northern Belize, distributed along a salinity gradient, are strongly phosphorus limited and dominated largely by three species of emergent macrophytes: Eleocharis cellulosa, Cladium jamaicense, and Typha domingensis. We assessed changes in root and sediment phosphatase activities of each species to simultaneous changes of nutrients (N, P) and salinity in a mesocosm experiment. Phosphorus and nitrogen treatment effects on both root and sediment phosphatase were highly significant for all the species, while salinity significantly affected root phosphatase activity in Cladium only. All species showed a significant negative correlation between root phosphatase activity and increasing tissue P content until a threshold of 0.2% P, 0.15% P and 0.12% P in Eleocharis, Cladium and Typha, respectively. There was also a significant negative correlation between soil available P and root and sediment phosphatases in all species. Activity of root phosphatases of Eleocharis and Typha were positively correlated with root tissue N. Both root and sediment phosphatases of all three species were positively correlated with soil available N. The strongest (positive) correlation was found between phoshatase activites and N/P ratios. The results confirmed that these systems are P-limited and that extracellular phosphatases respond to P enrichment by decreasing their activities. Expression of root phosphatase activity by dry root weight, sediment volume, or whole plant biomass gave very different relative results across nutrient treatments and species, suggesting that root phosphatase activities need to be interpreted in a wider context that considers root density.     

Tandem duplication of alkaline phosphatase genes and polymorphism in the intergenic sequence in<Emphasis Type="Italic"> Bombyx mori</Emphasis>

Alkaline phosphatases are ubiquitous in organisms from bacteria to human. Two alkaline phosphatase genes, Alp-m and Alp-s, were independently cloned from the silkworm Bombyx mori. They were mapped to a small DNA region and shown to be organized in tandem. Exon-intron structures of the two genes were highly conserved, with the exception of the second intron in Alp-m, which has no counterpart in Alp-s. The similarity between the nucleotide sequences of the exons of the two genes was strikingly high (60–79%), suggesting that Alp-m and Alp-s originated from a duplication of their common ancestor gene. The intergenic sequence between the two Alp genes shows length polymorphism in different B. mori strains, which can be explained by presence/absence of two putative insertion sequences. This structural variation suggests a possible scenario for the divergence of the two Alp genes after the duplication event.Communicated by G. Reuter     

Seasonal and environmental influences on antioxidative protection inPeridinium gatunense in Lake Kinneret

As the only freshwater lake in Israel, Lake Kinneret serves a number of important functions which directly rely upon the viability of the water. The annual outbreak of a dinoflagellate bloom strictly governs the nature of the macro and micro food web and ultimately determines water quality.The freshwater dinoflagellatePeridinium gatunense is subject to a wide range of environmental stresses throughout the spring bloom period. It was confirmed that SOD played an important antioxidative maintainance role throughout the bloom, especially during periods of relatively high photosynthetic activity (820 mg C m^–2 day^–1), when activity reached 500 Units SOD mg protein^–1. In addition, high light stress (>300 mol photons m^–2 s^–1) induced SOD activity, despite the low dissolved inorganic carbon (DIC) concentrations at the end of the bloom (1.3 mM). Catalase activity was only significant at the end of the bloom, peaking at 120 mol O₂ mg protein^–1 min^–1, when induced by photorespiratory activity.A series of experiments withPeridinium cultures showed that 2 × 10^–4 M ascorbate inhibited catalase activity >50% within 15 min incubation, bothin vivo andin vitro. It is suggested that the high concentrations of ascorbate, found previously inPeridinium during early and mid-bloom (0.2–1.6 mM), not only eliminate H₂O₂ build-up, but also prevent (directly or indirectly) the induction of catalase.     

Phosphate requirement of Anabaena oscillarioides and its ecological implications

Anabaena oscillarioides (pure culture isolated from Waikato River, lat. 38°S, long. 176°E, North Island, New Zealand) was shown to be phosphorous deficient with low internal cellular phosphours content and high induced alkaline phosphatase activity when it was grown in membrane-filtered river water in batch cultures under defined laboratory conditions. The calculated relatively high Ks (half-saturation substrate constant) for inorganic phosphate is 67 µg 1^–1, which is above usual river level for the nutrient. This explains the low density of the algae in the river where maximum growth is limited by low phosphorus concentration and the algae survive at suboptimal growth rate. Growth rate of the algae is proportional to phosphate concentration up to 100 µg 1^–1 beyond which increases in phosphate concentration only affects final yield. When the river was supplemented with 100 µg 1^–1 phosphate, the deficiency symptoms disappeared and the doubling time was reduced from two days to half a day.     

The role of Gloeotrichia echinulata in the transfer of phosphorus from sediments to water in Lake Erken

The abundance of Gloeotrichia echinulata colonies in the sediments of Lake Erken and their phosphorus content were investigated to determine the contribution of Gloeotrichia colonies to total sediment phosphorus. Moreover, the potential size of the algal inoculum and the migration to the water during summer were estimated.The surplus phosphorus content of the resting colonies in the sediment was about 45% of total phosphorus, which maximized at 8.5 µg P (mg dw)^–1 or 81 ng P colony^–1. The C:P ratio (by weight) in the early colonies appearing in the lake water was 50:1, while the ratio stabilized at 150 during the major migration period. The internal supply of surplus phosphorus was used during the pelagic growth of the colonies.The internal phosphorus loading to the epilimnion of Lake Erken due to Gloeotrichia migration could, from the measurements of the increase in particulate epilimnetic phosphorus, be estimated at 40 mg P m ^–2 or 2.5 mg P m^–2 d^–1 in late July and early August. Determination of the number of colonies in the sediment before and during the migration verified this value to be a conservative estimate of the internal phosphorus loading due to Gloeotrichia migration to the epilimnion in Lake Erken.The sediment P content calculated from the P concentration in early epilimnion colonies resulted in a value of 35 µg P (g dw)^–1 as a maximum. This corresponds to only 3% of the total phosphorus content in Lake Erken sediment.     

Phosphorus availability and alkaline phosphatase activities in two Israeli fishponds

1. Alkaline phosphatase activities and the release of orthophosphate from endogenous substrates by these enzymes were measured in waters from two commercial fishponds in the watershed area of Lake Kinneret (Northern Israel). These data were compared with results from the lake at seasons of adequate or limited phosphorus supply. In the fishponds, high Relative Phosphatase Activity ratios (>2) and relatively large amounts of orthophosphate extracted from plankton by autoclaving (average 27% of readily available phosphorus) indicated adequate, or even excess, levels of phosphorus availability despite elevated pond productivity (2 to 3 tons carp/ha/yr). Therefore, we suggest that decreasing routine phosphorus fertilization of these ponds would not affect overall productivity but would eventually lower the amounts of phosphorus reaching Lake Kinneret.
2. In general, the R. P. A. ratio may be a useful index to evaluate phosphorus availability for a wide range of natural waters. Values for this ratio of <1 and >2 appear indicative of limited or adequate phosphorus availability respectively.
3. Three sources of orthophosphate, (Pi), readily available to phytoplankton, are indicated: (1) enzymatically released Pi, (2) Pi in intracellular pools and (3) Pi initially present in the water. Although the first source is always important, relatively greater amounts of Pi are contributed by the other fractions in situations of plentiful phosphorus availability.
4. Activity of free dissolved phosphatases was found in filtered samples of fishpond water. However, neither these enzymes or added phosphatases released significant amounts of Pi from the dissolved organic phosphorus compounds in the filtered water.

     

Control of phosphorus loading and flushing as restoration methods for Lake Veluwe,The Netherlands

As a result of high nutrient loading Lake Veluwe suffered from an almost permanent bloom of the blue-green algaOscillatoria agardhii Gomont. In 1979, the phosphorus loading of the lake was reduced from approx. 3 to 1 g P.m^–2.a^–1. Moreover, since then the lake has been flushed during winter periods with water low in phosphorus. This measure aimed primarily at interrupting the continuous algal bloom. The results of these measures show a sharp decline of total-phosphorus values from 0.40–0.60 mg P.l^–1 (before 1980) to 0.10–0.20 mg P.l^–1 (after 1980). Summer values for chlorophylla dropped from 200–400 mg.m^–3 to 50–150 mg.m^–3.The increase in transparency of the lake water was relatively small, from summer values of 15–25 cm before the implementation of the measures to 25–45 cm afterwards. The disappointing transparency values may be explained by the decreasing chlorophylla and phosphorus content of the algae per unit biovolume. Blue-green algae are gradually loosing ground. In the summer of 1985 green algae and diatoms dominated the phytoplankton for the first time since almost 20 years. To achieve the ultimate water quality objectives (transparency values of more than 100 cm in summer), the phosphorus loading has to be reduced further.