The effect of phosphite on the sexual reproduction of some annual species of the jarrah (Eucalyptus marginata) forest of southwest Western Australia

Phosphite is a cost-effective fungicide used to control the pathogen Phytophthora cinnamomi which is damaging the diverse flora of the southwest of Western Australia. Three annual species of the southwest jarrah (Eucalyptus marginata) forest of Western Australia (Pterocheata paniculata, Podotheca gnaphalioides and Hyalosperma cotula), were studied to determine the effect of the fungicide phosphite on the species’ reproduction. Phosphite at concentrations of 2.5, 5 and 10 g L^–1 reduced pollen fertility of Pt. paniculata when plants were sprayed at the vegetative stage. Pollen fertility of all three species was reduced when plants were sprayed at anthesis with 10 g L^–1 phosphite. Seed germination was reduced by phosphite in Pt. paniculata and H. cotula when plants were sprayed in the vegetative stage. Phosphite when sprayed at anthesis at a concentration of 5 g L^–1 reduced seed germination of H. cotula. Phosphite at concentrations of 5 and 10 g L^–1 killed a proportion of plants from all three species and up to 90% of Po. gnaphalioides plants. The frequent application of phosphite, therefore, may reduce the abundance of annual plants in this ecosystem. Received: 14 December 2000 / Revision accepted: 10 March 2001     

Measurement of the barrier to inversion of configuration in acyclic phosphite triesters

Synthesis of three acyclic chiral phosphites is reported, in the form of dithymidine phosphite triesters. These diastereomerically pure P-stereogenic phosphites undergo epimerization at a measurable rate at 150 °C. When the alcohols on the deoxyribose moieties are protected as acyls, decomposition is minimized and by computer fitting, rate constants for epimerization can be extracted. These allow for the first time calculation of the barrier to inversion of configuration in phosphite triesters, giving ΔG^†(150 °C) = 33.0 ± 0.2 kcal mol⁻¹, comparable to the inversion barrier seen for phosphines.     

Alterations of alkaline phosphatase activity during adaptation of Escherichia coli to phosphite and hypophosphite

When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, they responded to this situation primarily in the same way as phosphatelimited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compklensatae for the shortage increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite or hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO ₃ ^3- or PO ₂ ^3- was completely abolished if the cells were again grown with PO ₄ ^3- as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO ₃ ^3- and PO ₂ ^3- , but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite.Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO ₄ ^3- to PO ₂ ^3- . Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

Mechanism and applications of phosphite dehydrogenase

Phosphite dehydrogenase catalyzes the NAD+-dependent oxidation of hydrogen phosphonate (common name phosphite) to phosphate in what amounts to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metabolism in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.     

Synthesis and characterization of an unusual equatorially substituted di-manganese compound. The first structural determination of a phosphite compound of type [Mn2(CO)9P]

Reaction of (acetonitrile)-nona(carbonyl)-di-manganese and tri-iso-propyl phosphite provided the new phosphite compound [Mn₂(CO)₉L]. The product was fully characterized and the molecular structure was determined by single crystal X-ray diffraction. This represents the first structural analysis of a di-manganese carbonyl compound containing phosphite ligands. In addition, this compound displays a rare example of an equatorially substituted di-manganese carbonyl compound.     

Effect of phosphate on the toxicity of phosphite in Phytophthora palmivora

The concentration of P_i in the growth medium had a marked effect on the amount of the fungicide phosphite accumulated by Phytophthora palmivora. The mass of mycelium, produced after 7 days growth in a medium containing excess P_i, was not markedly inhibited until phosphite concentrations of 10 mM or greater were supplied. In contrast, in a P_i-deficient medium, growth was inhibited by 0.1 mM phosphite. In the latter medium, the fungus took up significantly more phosphite once P_i had been depleted from the medium (from day 4 to 6). When 7 day old mycelium was presented with either 1 mM P_i or phosphite, the P_i-deficient mycelium transported either anion at almost eight times the rate found in the P_i-excess mycelium. The level of alkaline phosphatase present in the P_i-deficient mycelium was also significantly elevated.     

A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD⁺/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP⁺ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD⁺ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.     

Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

Anaerobic dissimilatory phosphite oxidation,an extremely efficient concept of microbial electron economy

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (−690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD⁺-dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO₂ fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.     

Reaction of a bulky phosphite with [Ru3(CO)12]: The molecular structure of one of the decomposition products

Mono- and bis-substituted phosphite complexes [Ru₃(CO)_12−x L_x] (L = tris(2,4-di-tert-butylphenyl) phosphite; x = 1, 2) were synthesized by simple substitution reactions, and were characterized by spectroscopic methods. The monosubstituted ruthenium complex disproportionates in acetone producing a mononuclear ruthenium complex as one of the decomposition products. Single crystal X-ray diffraction analysis established the molecular structure of this new compound.     

New enzyme cycling method for determination of oxidized and reduced nicotinamide adenine dinucleotide

A highly sensitive enzyme cycling method for the measurement of NAD⁺(H) concentration in tissue extracts has been described. The assay measured propanediol formed during the coupled oxidation-reduction reactions between ethanol and lactaldehyde as catalyzed by liver alcohol dehydrogenase in the presence of catalytic concentrations of NAD⁺(H). The assay can be used to determine NAD⁺(H) concentrations in the range of 0.4 to 4 pmoles of coenzyme in a 0.1 ml reaction volume.     

Enzymic method for the quantitative determination of reduced glutathione

A new specific and sensitive assay method for reduced glutathione has been developed. It is based on the reaction HCHO + NAD⁺ + H₂O → HCOOH + NADH + H⁺, catalyzed by formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC.1.2.1.1) in the presence of reduced glutathione. Oxidized glutathione and other thiols do not interfere in this reaction. A purification procedure for formaldehyde dehydrogenase from beef liver is presented.The influence of cysteine and some other thiols, leucine, ascorbate, lactate, pyruvate and four acids generally used for deproteinization is reported. The results obtained by this method from human blood and rat tissues are compared with those obtained by Ellman's method.     

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD⁺ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD⁺ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 × 10^?14 mol NADH or NAD⁺ per assay.     

Potassium phosphite affects growth,antioxidant enzymes activity and alleviates disease damage in cucumber plants inoculated with Pythium ultimum

In the present study, the impact of potassium phosphite on response of cucumber plants inoculated with Pythium ultimum var. ultimum was assessed. Variations in the accumulation of both antioxidant enzymes and growth parameters were investigated. The results revealed that fresh and dry weights of shoot and root exhibited up to 2 fold increase in potassium phosphite-treated plants. The concentrations of peroxidase, superoxide dismutase and catalase were significantly higher in the plants treated with 4 g L^?1 of potassium phosphite compared to other treatments, at 48 h after inoculation. It was demonstrated that both 2 and 4 g L^?1 treatments could alleviate the disease damage to a high extent, while control plants were severely damaged by the pathogen. The results of this study suggest that the increased induction of antioxidant enzymes (2.2, 2.8 and 4 fold increase for superoxide dismutase, catalase and peroxidase, respectively) might have alleviated damping-off symptoms leading to increased plant growth and yield.     

Hydrothermal synthesis, crystal structure and physical properties of a new gadolinium phosphite hydrate

A new compound Gd₄(H₂O)₅(HPO₃)₆ (1) was isolated from the reaction of gadolinium chloride GdCl₃ · nH₂O with H₃PO₃ under hydrothermal conditions. The crystal structure was solved by means of single-crystal X-ray diffraction. The structure is composed of parallel gadolinium-oxygen polyhedra chains connected by phosphite ions, and shows three-dimensional (3D) open-framework with eight-membered ring (8MR) channels. The structure is different from its counterpart Eu₂(H₂O)_2.5(PO₃H)₃ due to the effect of lanthanide contraction. The intensive intrinsic UV emission of this compound at λ_max = 312 nm comes from the spin-forbidden ⁶P_7/2 → ⁸S_7/2 f-f transition of the Gd³⁺ ions. No magnetic order was observed for this compound.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

A simple and sensitive assay for 9-hydroxyprostaglandin dehydrogenase

The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem.250, 2789] has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD⁺ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9-hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[9β-³H]PGF_2α is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent K_m for 15-keto-13,14-dihydro-PGF_2α is 0.66 μm. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous findings reported in the literature.     

Two indirect methods for detecting ureide synthesis by nodulated legumes

Two methods were developed for the detection of altered ureide metabolism in legume nodules. Both techniques are based on the positive correlation between the presence of high xanthine dehydrogenase (EC 1.2.1.37) specific activity in nodules and the ability of those nodules to produce the ureides, allantoin and allantoic acid. In the first method, nodulated legumes are treated for 2 weeks with a soil drench of allopurinol. After allopurinol treatment, leaves of N₂-fed, ureide-producing legumes, soybean, cowpea, and lima bean, became very chlorotic. Leaves of KNO₃⁻ or NH₄Cl-fed ureide-producing legumes were unaffected by the allopurinol treatment. Leaves of the amide-producing legumes, alfalfa, clover, peak, and lupin, were unaffected by the allopurinol treatment with N₂, KNO₃, or NH₄Cl as nitrogen source. These experiments showed that long-term allopurinol treatments are useful in differentiating between ureide- and amide-producing legumes when effectively nodulated. A second method was developed for the rapid, qualitative estimation of xanthine dehydrogenase activity in legume nodules. This method utilizes pterin, an alternate substrate for xanthine dehydrogenase. Xanthine dehydrogenase hydroxylates pterin in the presence of NAD⁺ to produce isoxanthopterin. When exposed to long wave ultraviolet light (365 nanometers), isoxanthopterin emits blue fluorescence. When nodules of ureide-producing legumes were sliced in half and placed in microtiter plate wells containing NAD⁺ and pterin, isoxanthopterin was observed after 6 hours of incubation at room temperature. Allopurinol prevented isoxanthopterin production. When slices of amide-producing legume nodules were placed in wells with pterin and NAD⁺, no blue fluorescence was observed. The production of NADH by xanthine dehydrogenase does not interfere with the fluorescence of isoxanthopterin. These observations agree with the high specific activity of xanthine dehydrogenase in nodules of ureide-producing legumes and the low activity measured in amide-producing nodules. The wild soybean, Glycine soja Sieb. and Zucc., was examined for ureide synthesis. Stems of wild soybean plants had a high ureide abundance with N₂ as sole nitrogen source when nodulated with either Rhizobium fredii or Bradyrhizobium japonicum. Ureide abundance declined when nitrate or ammonium was added to the nutrient solution. Nodule slices of these plants produced isoxanthopterin when incubated with pterin. Nodule crude extracts of G. soja had high levels of xanthine dehydrogenase activity. Both Glycine max and G. soja plants were found to produce ureides when plants were inoculated with fast-growing R. fredii. The two methods described here can be used to discriminate ureide producers from amide producers as well as detect nitrogen-fixing legumes which have altered ureide metabolism. A nodulated legume that lacks xanthine dehydrogenase activity as demonstrated by the pterin assay cannot produce ureides since ureide synthesis has been shown to require xanthine dehydrogenase activity both in vivo and in vitro. A nodulated legume that remains green during allopurinol treatment also lacks ureide synthesis since the leaves of ureide-producing legumes are very chlorotic following allopurinol treatment.     

A fluorimetric method for continuously assaying ATPase: application to small specimens of glycerol-extracted muscle fibers.

A continuous fluorimetric method using auxiliary-coupling enzymes such as pyruvate kinase and lactate dehydrogenase for measuring ADP production to assay ATPase activity is described. This method is simpler, more rapid, and more sensitive than the previously used spectrophotometric method. The application of this method for studying the ATPase of rabbit psoas muscle fibers during Mg²⁺-ATP activation is also illustrated and discussed.