Comparative Study of Uptake and Cellular Distribution of Hg203-Labeled Phenyl-Mercuric Acetate and Mercuric Acetate by Pea Roots

Uptake and cellular distribution of mercury²⁰³ from dilute mercuric acetate or phenylmercuric acetate solutions by excised pea roots (Pisum sativum) have been investigated. The time course of uptake showed that the amount of mercury uptake was increased with the time of incubation, and was similar for inorganic mercury or phenylmercuric acetate. The trend of mercury²⁰³ incorporation into cellular components from mercuric acetate and phenylmercuric acetate differed greatly as the time of incubation increased. The concentrations of mercuric acetate and phenylmercuric acetate solutions or the temperature of incubation also affected the mercury²⁰³ uptake as well as its cellular distribution. Longer time of exposure or higher concentration resulted in a greater mercury incorporation into mitochondrial fraction from phenylmercuric acetate than from inorganic mercury. This difference in intracellular distribution may be responsible for the degree of toxicity between inorganic mercury and phenylmercuric acetate in biological systems.     

Inhibition of chloroplast reactions with phenylmercuric acetate

Phenylmercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 μm concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 μm phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 μm phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 μm phenylmercuric acetate and very much higher cooncentrations—500 μm—are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)-1,1-dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer.     

Stimulatory effect of phenylmercuric acetate and benzene on the growth of a broad spectrum mercury-resistant strain of Bacillus pasteurii

A broad spectrum mercury-resistant organism, Bacillus pasteurii DR₂, was isolated from the effluents of Durgapur Steel Plant, India. In the presence of phenylmercuric acetate or benzene the rate of glucose uptake and the level of some metabolic enzymes increased significantly. Inhibition of the glucose oxidation rate by these chemicals in lysozyme-treated cells indicated that these compounds facilitated the transport of glucose across the cell wall and thereby stimulated growth.     

A reappraisal of the reaction of butyrylcoenzyme A dehydrogenase with phenylmercuric acetate. Evidence that de-greening involves a reaction of the tightly bound thioester

Phenylmercuric acetate reversibly de-greens butyryl-CoA dehydrogenase from Megasphaera elsdenii, abolishing the absorption band at 710nm. The view that this is a result of modification of a protein thiol group is re-examined in the light of the following new observations. (i) After treatment with phenylmercuric acetate, the enzyme's ability to be re-greened by addition of thiols was not decreased by gel filtration or precipitation with (NH(4))(2)SO(4). (ii) Phenylmercuric acetate caused the same extent of de-greening whether added in a few large amounts or many small ones. The overall time taken for de-greening was, however, greatly extended when many small additions were made. (iii) In Tris/acetate buffer, pH7.5, 3.5mol of phenylmercuric acetate/mol of enzyme subunit was required for complete de-greening, compared with only 2.5mol/mol in phosphate buffer, pH7. (iv) None of the groups that react with phenylmercuric acetate is accessible to iodoacetate or iodoacetamide. (v) On a molar basis dithiothreitol, mercaptoethanol and CoA are equally effective in re-greening the enzyme. (vi) Provided that phenylmercuric acetate is not present in excess, the de-greened enzyme forms normal and stable complexes with crotonyl-CoA and acetoacetyl-CoA. (vii) When a small excess of phenylmercuric acetate is present, full stable development of the enzyme-acetoacetyl-CoA complex requires addition of several mol of acetoacetyl-CoA/mol of enzyme subunit. (viii) The ability of de-greened enzyme to be immediately re-greened by an excess of thiol declines with time, more rapidly at pH6 than at pH7 or 8, but at all three pH values the instantaneous re-greening was followed by a slow phase of further increase in A(710). This further recovery was most extensive and most rapid at pH8. These findings are reminiscent of the previously described reversible decline in the re-greening capacity of a protein-free acid extract of green butyryl-CoA dehydrogenase. It is concluded that the likely cause of de-greening is chemical modification of the tightly bound thioester rather than a protein thiol group. The reversibility would be explained if the thioester exists on the surface of the enzyme in equilibrium with free CoA and a lactone, or if the acyl group is readily and reversibly transferred from the thiol of CoA to a protein side chain.     

Biodegradation of Phenylmercuric Acetate by Mercury-Resistant Bacteria

Selected cultures of mercury-resistant bacteria degrade the fungicide-slimicide phenylmercuric acetate. By means of a closed system incorporating a flameless atomic absorption spectrophotometer and a vapor phase chromatograph, it was demonstrated that elemental mercury vapor and benzene were products of phenylmercuric acetate degradation.     

Two electrogenic mechanisms contributing to the 560 nm absorption changes in intact Bryopsis chloroplasts

Light -induced absorbance changes at 560 nm in dark-adapted intact chloroplasts of the green alga, Bryopsis maxima were studied in the time range of 200 ms. The initial rise of the 560 nm signals constists of two major components which are both electrochromic absorbance changes of the carotenoids, sipnonein and/or siphonaxanthin, but different in mechanisms of the field formation.The first component (component S) is related to electron transport since it was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and showed a light-intensity dependence similar to that of electron transport in chloroplasts. In the presence of DCMU, component S could be restored on addition of proton-transporting electron donors such as reduced 2,6-dichlorophenol indophenol and phenazine methosulfate, but not on addition of N,N,N′,N′-tetramethyl-p-phenylenediamine which does not carry protons with electrons (Trebst, A. (1974) Annu. Rev. Plant Physiol. 25, 423–458). We propose that component S is due to the electric field set up by the proton translocation across the thylakoid membrane.The second component (component R) was resistant to DCMU and DBMIB. The light-intensity dependency of component R was similar to that of cytochrome f photooxidation which showed saturation at a relatively low light intensity. The magnitude of component R was markedly reduced by phenylmercuric acetate, suggesting the participation of ferredoxin and ferredoxin-NADP oxidoreductase in the mechanism of the field formation responsible for this component. In the presence of DCMU and phenylmercuric acetate, time courses of the 560 nm changes paralleled those of cytochrome f changes. These results indicate that component R is due to the electric field formed between oxidized cytochrome f and other intersystem electron carriers located in the inner part of the thylakoid membrane and reduced electron acceptors of Photosystem I situated on the membrane surface.The complex natures of the 560 nm changes, as well as the contributions of Photosystems I and II to the absorbance changes, are explained in terms of the two electrogenic mechanisms.     

Effects of metabolic poisons on rice: the comparative sensitivity of aerobic and anaerobic modes of germination

Rice germinates equally well when incubated in air and in nitrogen. It was therefore chosen for a comparative study of the effect of oxygen status in a single organism upon the activity of conventional metabolic poisons. Inhibitor activity was based upon the concentration required for 50% inhibition of germination. The inhibitors were: AgNO₃, HgCl₂, phenylmercuric acetate, iodoacetamide, KCN, NaN₃, NaF, fluoroacetate, 2,4-dinitrophenol, Na₂HAsO₄ and CO. Only 5 inhibitors out of 11 differed significantly in their effects on aerobic and anaerobic germination. These included phenylmercuric acetate (N₂), sodium fluoroacetate (air), NaN₃ (air), and 2,4-dinitrophenol (N₂). CO activity was manifest only in air and it was photo-reversible. The effects of CO, NaN₃, and fluoroacetate were consistent with their conventional role in aerobic metabolism. The failure of KCN to discriminate was attributed to its relative non-specificity. 2,4-Dinitrophenol behaved anomalously, requiring a 20-fold lower concentration for inhibition under nitrogen than under air. Among sulfhydryl poisons, only phenylmercuric acetate differentiated between germination in air and nitrogen and was nearly 8 times more active under nitrogen. Uptake measurements using phenylmercuric acetate and arsenate showed both compounds to be in greater concentration under aerobic conditions, thereby rendering unlikely any explanation based upon greater permeability under nitrogen. In addition to other anomalies, the fact that NaN₃, fluoroacetate and CO inhibited anaerobic germination at any concentration requires explanation. It was concluded that the general significance and utility of metabolic inhibitor studies in vivo required further evaluation.     

Translocatable resistance to mercuric and phenylmercuric ions in soil bacteria.

Of a sample of 42 gram-negative Hg-resistant bacteria, three (a Pseudomonas fluorescens, a Klebsiella sp. and a Citrobacter sp.) contained translocatable elements conferring resistance to Hg2+ (all three) and to Hg2+ and phenylmercuric acetate (P. fluorescens). The discovery of transposable phenylmercuric acetate resistance extends the range of known resistance "transposons" from heavy metals and antibiotics to organometallic compounds.     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

A kinetic study of phosphorus absorption by excised maize roots from flowing solutions influenced by 2,4 dinitrophenol and viscosity of solution

The effect of 2,4 dinitrophenol and increased viscosity of the absorption solution on the absorption of phosphorus by excised roots of maize plants was investigated. The concentration of the solution was 0.1 mM KH₂PO₄, the activity of³²P was 52 µCi l^-1. The temperature of the absorption solution was 26 °C, pH 5.5, aeration prior to the experiment. There was 11 of solution for every 1 g of roots. Two basic variants were used for comparison: with non-flowing solution and with solution flow (circulation) of 0.162 cm s^-1, respectively. In all cases, 2,4 dinitrophenol reduced the rate of phosphorus absorption by the roots regardless of the mechanism of phosphorus supply to the roots (diffusion, mass flow). If it is proved that 2,4 dinitrophenol inhibits the active uptake of phosphorus, then the uptake of phosphorus by the roots increased under the influence of mass flow will be active,i.e., connected with energy metabolism. Raising the viscosity of the absorption solution 3.3 times over that of water by means of potato starch substantially reduced the absorption of the phosphorus transported to the absorption zone by diffusion and practically did not affect the rate of absorption, or the amount of anions transported to the absorption area by mass flow.     

Root architecture of Arabidopsis is affected by competition with neighbouring plants

How roots detect and respond to the presence of neighbors is relevant to understand plant belowground interactions. The aim of the present work was to evaluate the effect of the presence of neighboring plants and the limited availability of phosphorus on root architecture. A target plant of Arabidopsis thaliana (Ler or Col) was surrounded by combinations of two individuals (Ler and Col), and subjected to different growth conditions (levels of activated charcoal (AC) and phosphorus). Both accessions consistently concentrated their roots towards the competition zone shared with a neighbor of the same accession, avoiding the side shared with the other accession. All these competition strategies disappeared when plants were limited by phosphorus or when activated charcoal was added to the growth media. Plants produced consistently fewer but longer lateral roots when activated charcoal was added to the growth media irrespective of the neighbors. Our results indicate a direct role of secondary metabolites present in the root exudates and phosphorus availability in the response of presence and identity of neighboring roots.     

Physiological and cellular mechanisms of natural herbicide resource from Aglaia odorata Lour. on bioassay plants

A bioassay-guided fractionation of the allelochemicals of the ethyl acetate fraction extracted from Aglaia odorata led to isolation of a bisamide, odorine. The growth inhibitory effects of odorine and ethyl acetate fraction were studied for comparison on Echinochloa crus-galli. Odorine and ethyl acetate fraction of A. odorata could inhibit the germination and seedling growth of E. crus-galli, with ethyl acetate fraction being more potent. Thus, ethyl acetate fraction was selected for further experiment, E. crus-galli seeds were studied the effects of the ethyl acetate fraction from A. odorata leaves in wettable powder formulation on imbibition and ??-amylase activity. It was found that treated seeds showed lower imbibition and ??-amylase activity. The results of cytogenetic bioassay in Allium cepa roots showed that ethyl acetate fraction inhibited cell mitosis and induced mitotic abnormalities resulting from its action on chromatin organization and mitotic spindle in the exposed roots.     

Architectural Tradeoffs between Adventitious and Basal Roots for Phosphorus Acquisition

Adventitious rooting contributes to efficient phosphorus acquisition by enhancing topsoil foraging. However, metabolic investment in adventitious roots may retard the development of other root classes such as basal roots, which are also important for phosphorus acquisition. In this study we quantitatively assessed the potential effects of adventitious rooting on basal root growth and whole plant phosphorus acquisition in young bean plants. The geometric simulation model SimRoot was used to dynamically model root systems with varying architecture and C availability growing for 21 days at 3 planting depths in 3 soil types with contrasting nutrient mobility. Simulated root architectures, tradeoffs between adventitious and basal root growth, and phosphorus acquisition were validated with empirical measurements. Phosphorus acquisition and phosphorus acquisition efficiency (defined as mol phosphorus acquired per mol C allocated to roots) were estimated for plants growing in soil in which phosphorus availability was uniform with depth or was greatest in the topsoil, as occurs in most natural soils. Phosphorus acquisition and acquisition efficiency increased with increasing allocation to adventitious roots in stratified soil, due to increased phosphorus depletion of surface soil. In uniform soil, increased adventitious rooting decreased phosphorus acquisition by reducing the growth of lateral roots arising from the tap root and basal roots. The benefit of adventitious roots for phosphorus acquisition was dependent on the specific respiration rate of adventitious roots as well as on whether overall C allocation to root growth was increased, as occurs in plants under phosphorus stress, or was lower, as observed in unstressed plants. In stratified soil, adventitious rooting reduced the growth of tap and basal lateral roots, yet phosphorus acquisition increased by up to 10% when total C allocation to roots was high and adventitious root respiration was similar to that in basal roots. With C allocation to roots decreased by 38%, adventitious roots still increased phosphorus acquisition by 5%. Allocation to adventitious roots enhanced phosphorus acquisition and efficiency as long as the specific respiration of adventitious roots was similar to that of basal roots and less than twice that of tap roots. When adventitious roots were assigned greater specific respiration rates, increased adventitious rooting reduced phosphorus acquisition and efficiency by diverting carbohydrate from other root types. Varying the phosphorus diffusion coefficient to reflect varying mobilities in different soil types had little effect on the value of adventitious rooting for phosphorus acquisition. Adventitious roots benefited plants regardless of basal root growth angle. Seed planting depth only affected phosphorus uptake and efficiency when seed was planted below the high phosphorus surface stratum. Our results confirm the importance of root respiration in nutrient foraging strategies, and demonstrate functional tradeoffs among distinct components of the root system. These results will be useful in developing ideotypes for more nutrient efficient crops.     

Nutrient removal from on-site domestic wastewater in horizontal subsurface flow reed beds in Ireland

In order to investigate the nutrient (namely nitrogen and phosphorus) removal efficiency and the governing internal dynamics of the most widely used wetland type, the horizontal subsurface flow reed bed, in receiving domestic septic tank and secondary effluent in a temperate climate such as Ireland, two systems were designed, constructed and rigorously monitored for a period of over 2 years. Nitrogen removal, as expected, was found to be poor across both reed beds, with only 29% removal of TN across the secondary treatment bed and 41% removal across the tertiary treatment bed, with little distinctive seasonal change. A ¹⁵N stable isotope tracer study revealed, in line with the results from the chemical analysis, that nitrogen kinetics in the secondary treatment bed were dominated by continuous plant litter decomposition and mineralisation processes converting stored org-N to NH₄-N indefinitely. Similar analysis on the tertiary treatment bed indicated that only limited denitrification of the oxidized forms of N was occurring in the anoxic environment of the bed, while NH₄-N and org-N were merely changing form on a cyclic basis. Removal of PO₄-P from the secondary and tertiary treatment beds was equally poor at rates of 45% and 22%, respectively. While at their maximum growth in the third year of operation, the total phosphorus in the stems and roots of the Phragmites australis in the secondary treatment bed equated to only 10% of the total P removed over the duration of the bed's operation. In the tertiary treatment bed, more seasonal variability was recorded with intermittent negative removal found during winter periods. This was somewhat more reflected in the P-uptake study for this bed with the roots and stems of the Typha and Iris containing phosphorus, which accounted for 31% of the overall mass removed.     

Effect of adsorbed cations on phosphorus uptake by excised roots

Pretreatment of excised roots of Hordeum vulgare, Zea mays, and Glycine max with various salt solutions affected their subsequent rate of phosphorus absorption from 2 × 10⁻⁵m KH₂PO₄. The rate of absorption was greatest for roots pretreated with trivalent cations, intermediate with divalent cations and lowest with monovalent cations. It appeared that the pretreatment involved a rapid exchange reaction at the root surface which was reversible. A 1 min pretreatment was effective for more than 20 min. The acceleration of phosphorus uptake by roots produced by polyvalent cations may be due partly or entirely to a greater reduction in the electrical potential at the root surface or within the pores of the negatively charged cell wall by polyvalent cations than by monovalent cations.     

Phosphorus absorption by intact maize plants from flowing solutions influenced by 2.4-dinitrophenol and viscosity of solution

The investigation of phosphorus absorption by intact plants during a short period has above all confirmed the validity of the results obtained in the foregoing study of the kinetics of absorption by excised roots. Further, the results show the unquestionably important role of mass flow in transporting ions to plant roots, mainly at lower and medium concentrations, that is, from about 0.1 to 10 mM. Under conditions of growth close to the optimum, the supply by means of mass flow can be sufficient even at lower concentrations of phosphorus, such as 1.47 mM KH₂PO₄, or the absorption of phosphorus by plants can be higher than in the case of ions being transported to roots by diffusion. With a higher absorption the phosphorus distribution somewhat changes as well, relatively more of it being accumulated in the roots. 2.4-DNP applied to the nutrient solution at a concentration of 10^-5 M reduces the phosphorus absorption.     

Evidence against an intermediary role of abscisic acid in stomatal closure induced by phenylmercuric acetate and faresol

The hypothesis was tested that stomatal closure induced by the antitranspirants phenylmercuric acetate and farnesol is mediated by an increase in abscisic acid content. Isolated leaves of Spinacia oleracea L. cv. Müma, Tradescantia X andersoniana Ludw. et Rohw. and Commelina communis L. were incubated in solutions of phenylmercuric acetate (10⁻³ M ) and of farnesol (2 × 10⁻³ M ). In all three species, a reduction in stomatal aperture was observed. The extent and velocity of the reaction differed between the species and also between the two compounds applied. The abscisic acid content of the leaves was determined after stomatal closure had been achieved. No significant increase in abscisic acid level was found in treated leaves of Spinacia and Commelina . In Tradescantia , on the contrary, a reduction to about 50% was observed after 120 min treatment. Visible damage of the treated leaves and membrane alterations observable by electron microscopy occurred, especially after treatment with farnesol. These changes in membrane structure suggest a connection with the reduction in stomatal aperture.     

Effect of Temperature on the Radial Exchange of Labelled Water in Maize Roots

The temperature dependence of the efflux kinetics of labelledwater in isolated maize roots has been studied. The purposeof these experiments was to obtain the energy of activation,E (kcal/mole), of the rate-limiting step in this radial exchangeprocess under various experimental conditions. Estimates ofE were obtained from linear relations between ln{D'_w} and thereciprocal of the absolute temperature; values of the apparentdiffusion coefficient, D'_w, of labelled water in the root werefound from an analytical treatment of the efflux data in termsof a cylindrical diffusion model. The energy of activation forlabelled-water exchange in normal roots was 14.9 kcal/mole.The corresponding value for ‘dead’ (boiled) rootswas 3.9 kcal/mole. These values of E substantiate the view thatin normal roots the penetration of water across the membranesof the root cells constitutes the rate-determining step in theefflux whereas in ‘dead’ roots extracellular diffusionof water is the source of rate-control. Similar temperature dependence studies were performed on theefflux kinetics from normal and ‘dead’ roots treatedwith 10^–5 M phenylmercuric acetate (PMA). The energiesof activation for labelled-water exchange in normal and ‘dead’roots under these conditions were respectively 15.5 and 5.3kcal/mole. Moreover, the results of the efflux experiments onPMA-treated roots were considered to indicate that this inhibitorproduces an alteration in some structural aspect of the rate-controlling‘membranes’.     

High resolution microanalysis for phosphorus in Golgi complex beads of insect fat body tissue by electron spectroscopic imaging

Golgi complex beads are 10 nm particles arranged in rings on the smooth forming face of the Golgi complex that stain specifically with bismuth in arthropod cells. In vitro experiments with biological molecules spotted on to cellulose acetate strips indicated that bismuth bound to the beads through phosphate groups. We could detect a weak phosphorus signal from the beads using a new technique called electron spectroscopic imaging that is capable of very high spatial resolution (0.3–0.5 nm) and sensitivity (50 atoms of phosphorus). Detection was not obscured by tissue staining with bismuth or uranyl acetate or by using an inorganic buffer (Na cacodylate). Localization of phosphorus was greatly improved by using colour-enhanced computer pictures of the electron spectroscopic images and quantitating the images. The results indicate that the phosphorus content of the beads is large enough to account for their bismuth reactivity.     

Variations in leaf and root stoichiometry of Nitraria tangutorum along aridity gradients in the Hexi Corridor,northwest China

Nitrogen (N) and phosphorus (P) concentrations and N: P ratios between leaves and roots of Nitraria tangutorum along aridity gradients were studied. N. tangutorum was relatively limited by N in April (mean leaf N: P ratio = 11.13) and by P in August (mean leaf N: P ratio = 38.78). N and P in both leaves and roots were highly correlated across sampling sites. In April both leaf and root N and P concentrations increased along aridity gradients. Mean leaf N: P ratios changed slightly, but mean root N: P ratios increased with increasing aridity gradients. We suggest that leaf N: P ratios can indicate nutrient status at different plant growth stages, and root N: P ratios can signify if the amount of soil nutrients is insufficient.