Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Calcium-dependent zinc efflux in human red blood cells

Summary Zinc efflux from human red blood cells is largely brought about by a saturable mechanism that depends upon extracellular Ca²⁺ ions. It has aV _max of about 35 mol/10¹³ cells hr, aK _m for external Ca²⁺ of 1×10^–4 m, and aK _m for internal Zn²⁺ of 1×10^–9 m. External Zn²⁺ inhibits with aK _0.5 of 3×10^–6 m. Sr²⁺ is a substitute for external Ca²⁺, but changes in monovalent anions or cations have little effect on the Zn²⁺ efflux mechanism. It is unaffected by most inhibitors of red cell transport systems, although amiloride and D-600 (methoxyverapamil, a Ca²⁺ channel blocker) are weakly inhibitory. The transport is capable of bringing about the net efflux of Zn²⁺, against an electrochemical gradient, provided Ca²⁺ is present externally. This suggests it may be a Zn²⁺:Ca²⁺ exchange, which would be able to catalyze the uphill movement of Zn²⁺ at the expense of an inward Ca²⁺ gradient, which is it self maintained by the Ca²⁺ pump.     

Zinc-phosphorus interactions in two cultivars of tomato (Lycopersicon esculentum L.) grown in chelator-buffered nutrient solutions

Zinc-phosphorus interactions have been frequently studied using a diverse number of crop species, but attainment of reproducible Zn deficiencies, especially severe ones, has been hampered by the use of conventional hydroponic solutions wherein contaminating levels of Zn are often near-adequate for normal growth. We utilized novel, chelator-buffered nutrient solutions for precise imposition of Zn deficiencies. Tomato (Lycopersicon esculentum L. cv. Jackpot or Celebrity) seedlings were grown for 15 to 18 d in nutrient solutions containing 200, 600, or 1200 M P, and 0 to 91 M total Zn. Computed free Zn²⁺ activities were buffered at 10^-10.3 M by inclusion of a 100-M excess (above the sum of the micronutrient metal concentrations) of the chelator DTPA. At total added Zn=0, acute Zn deficiency resulted in zero growth after seedling transfer, and plant death prior to termination. Free Zn²⁺ activities 10^-10.6 M resulted in Zn deficiencies ranging from mild to severe, but activities 10^-11.2 were required to cause hyperaccumulation of shoot P to potentially toxic levels. Despite severe Zn deficiency (i.e. ca. 20% of control growth), tissue Zn levels were usually much higher than the widely reported critical value of 20 mg kg^-1, which may be an artifact of the selection of DTPA for buffering free Zn²⁺. Across Zn treatments, increasing solution P depressed growth slightly, especially in Celebrity, but corresponding increases in tissue P (indicative of enhanced P toxicity) or decreases in tissue Zn (P-induced Zn deficiency) were not observed. The depressive effect of P was also not explained by reductions in the water-soluble Zn fraction. Within 40 h, restoration of Zn supply did not ameliorate high leakage rates (as measured by K⁺ efflux) of Zn-deficient roots. Similarly, transfer of Zn-sufficient plants to deficient solutions did not induce leakiness within 40 h. Foliar sprays of ZnSO₄ almost completely corrected both Zn deficiency and membrane leakiness of plants grown in low-Zn solutions. Hence, maintenance of root membrane integrity appears to depend on the overall Zn nutritional status of the plant, and not on the presence of certain free Zn²⁺ levels in the root apoplasm.     

Competitive adsorption of Pb, Cd and Zn ions onto Eichhornia crassipes in binary and ternary systems

A batch sorption technique was used to study the biosorption of Pb²⁺, Cd²⁺ and Zn²⁺ ions onto the vastly abundant water hyacinth weed, Eichhornia crassipes biomass in binary and ternary systems at a temperature of 30 °C and pH 4.84. Mutual interference effects were probed using equilibrium adsorption capacity ratios, , where the prime indicates the presence of one or two other metal ions. The combined action of the metals was found to be antagonistic, and the metal sorption followed the order Pb²⁺  Cd²⁺  Zn²⁺. The behaviour of competitive biosorption for Pb–Cd and Pb–Zn combinations were successfully described by the Langmuir Competitive Model (CLM), whilst the model showed poor fitting to the Cd–Zn data. In conclusion, Pb²⁺ ions could still be effectively removed from aqueous solution in the presence of both Cd²⁺ and Zn²⁺ ions, but removal of the Cd²⁺ and Zn²⁺ ions would be suppressed in the presence of Pb²⁺.     

Continuous measurement of calcium influx in mammalian nonmyelinated nerve fibers: Effects of Na o,Ca o,and electrical activity

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K⁺ passes mainly through the inward rectifier potassium channels.Inhibition of K⁺ efflux by external Zn²⁺ (0.25 to 15mm) differs in three significant ways from inhibition by Ba²⁺. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn²⁺-sensitivity of K⁺ efflux does not depend on [K⁺] _o at constant internal potential. (3) Zn²⁺ inhibition is reduced by hydrogen ions, while Ba²⁺ inhibition is unaffected. Further, the Ba²⁺-sensitivity of K⁺ efflux is not altered by a half-inhibiting Zn²⁺ concentration, suggesting that the two ions do not interact at a common site.The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn²⁺ inhibition. After DEPC treatment Zn²⁺ inhibition is further reduced by low pH. DEPC has little effect on Ba²⁺ inhibition. Zn²⁺ inhibition is not altered by treatment with the sulfhydryl reagents 5,5-dithio-bis(2-nitrobenzoic acid) or dithiothreitol.The results can be described by either of two models in which two sites can bind Zn²⁺ and one or both of the sites may also bind H⁺. When both sites bind Zn²⁺, K⁺ efflux is inhibited, and a third site may then bind H⁺. The effects of DEPC can be accounted for by a decrease in H⁺ affinity of the first two sites by a factor of 50, and a decrease in Zn²⁺ affinity of these sites and of the H⁺ affinity of the third site by about one order of magnitude.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Ion efflux systems involved in bacterial metal resistances

Summary Studying metal ion resistances gives us important insights into environmental processes and provides an understanding of basic living processes. This review concentrates on bacterial efflux systems for inorganic metal cations and anions, which have generally been found as resistance systems from bacteria isolated from metal-polluted environments. The protein products of the genes involved are sometimes prototypes of new families of proteins or of important new branches of known families. Sometimes, a group of related proteins (and presumedly the underlying physiological function) has still to be defined. For example, the efflux of the inorganic metal anion arsenite is mediated by a membrane protein which functions alone in Gram-positive bacteria, but which requires an additional ATPase subunit in some Gram-negative bacteria. Resistance to Cd²⁺ and Zn²⁺ in Gram-positive bacteria is the result of a P-type efflux ATPase which is related to the copper transport P-type ATPases of bacteria and humans (defective in the human hereditary diseases Menkes' syndrome and Wilson's disease). In contrast, resistance to Zn²⁺, Ni²⁺, Co²⁺ and Cd²⁺ in Gram-negative bacteria is based on the action of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is called RND). Another new protein family, named CDF for cation diffusion facilitator has as prototype the protein CzcD, which is a regulatory component of a cobalt-zinc-cadmium resistance determinant in the Gram-negative bacteriumAlcaligenes eutrophus. A family for the ChrA chromate resistance system in Gram-negative bacteria has still to be defined.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Zinc and lead uptake by mycelium and regenerating protoplasts of Verticillium marquandii

The ability of the filamentous fungus Verticillium marquandii for Zn²⁺ and Pb²⁺ uptake from aqueous solution was studied. The 24-h-old living mycelium bound Zn²⁺ and Pb²⁺ (206.2 and 324.5 mg/g dry weight, respectively) effectively, in contrast to a very low Zn²⁺ uptake by autoclaved mycelium (20.2 mg/g). The most effective results were noted when the metals were introduced as acetates and incubated with mycelium for 24 h in case of Zn²⁺ while Pb²⁺ achieved the maximum level of metal binding after as early as 3 h. The cell wall was the main site of effective Zn²⁺ and Pb²⁺ binding by V. marquandii mycelium (91.0–93.6% of metals were located in cell wall after 24 h of exposure). The metabolic inhibitors: antimycin A and sodium azide had a strong limitation effect on Zn²⁺ uptake by a 24-h-old living mycelium, whereas Pb²⁺ binding did not decrease to a large extent. The freshly obtained protoplasts accumulated Zn²⁺ and Pb²⁺ on a low level in comparison with cells at different stages of cell wall regeneration. The use of regenerating protoplasts showed that resynthesis of cell wall was necessary for high binding of Zn²⁺, whereas Pb²⁺ uptake on the significant level took place during cell wall regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.     

EFFECT OF ZINC AVAILABILITY ON GROWTH,MORPHOLOGY, AND NUTRIENT INCORPORATION IN A COASTAL AND AN OCEANIC DIATOM1

We investigated the effect of Zn availability on growth rate (μ), cell morphology, and elemental stoichiometry and incorporation rate in two marine diatoms. For the coastal diatom Skeletonema costatum (Grev.) Cleve, the half‐saturation constant (K_S) for growth was 4.1 pM Zn²⁺, and growth ceased at ≤ 2.6 pM Zn²⁺, whereas for the oceanic diatom Thalassiosira oceanica Hasle, K_S was 0.5 pM Zn²⁺, and μ remained at ～40%μ_max even at 0.3 pM Zn²⁺. Under Zn‐limiting (Zn‐L) conditions, S. costatum decreased cell size significantly, leading to an 80% increase in surface area to volume ratio (SA/V) at Zn²⁺ of 3.5 pM compared to Zn‐replete (Zn‐R) conditions (at Zn²⁺ of 13.2 pM), whereas T. oceanica’s morphology did not change appreciably. Cell quotas of C, N, P, Si, and chl a significantly decreased under Zn limitation in S. costatum (at Zn²⁺ of 3.5 pM), whereas Zn limitation in T. oceanica (at Zn²⁺ of 0.3 pM) had little effect on quotas. Elemental stoichiometry was ～85C:10N:9Si:1P and 81C:9N:5Si:1P for S. costatum, and 66C:5N:2Si:1P and 52C:6N:2Si:1P for T. oceanica, under Zn‐R and Zn‐L conditions, respectively. Incorporation rates of all elements were significantly reduced under Zn limitation for both diatoms, but particularly for Si in S. costatum, and for C in T. oceanica, despite its apparent tolerance of low Zn conditions. With [Zn²⁺] in some parts of the ocean being of the same order (～0.2 to 2 pM) as our low Zn conditions for T. oceanica, our results support the hypothesis that in situ growth and C acquisition may be limited by Zn in some oceanic species.     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

Zn buffering capacity and Zn accumulation in Swiss chard for some sludge-amended soils

Zinc sorption by soils can greatly affect its availability to plants. This study was conducted to determine the relationship between the Zn sorption capacity and plant Zn accumulation in five sludge-amended soils using Swiss chard (Beta vulgaris L.) as an indicator plant. Zinc sorption as a function of Zn concentration and pH was determined for the soils which received no sludge amendment; also DTPA (diethylenetriaminepentaacetic acid) extractable Zn was determined in all soils. Whereas the responses of DTPA-Zn and plant Zn to pH and the quantities of Zn sorbed were similar, the logarithm of DTPA-Zn accounted for only 82% of the variability in the logarithm of Zn accumulation by the plants. The variability was better explained when pH was included with DTPA-Zn in stepwise multiple regressions. The Zn buffering capacity, defined as the ratio of the change in quantity of Zn sorbed ( Zn_s) to the change in Zn solution concentration (Zn₁) (or Zn_s/Zn₁), and the estimated quantity of Zn sorbed were used as a basis to measure Zn intensity. Zinc intensity, which reflects Zn solution concentration, was the predominant factor controlling Zn accumulation by Swiss chard, judging from the good fit of the values of both parameters to the Michaelis-Menten equation. The maximum Zn accumulation was approximately 9 mmol kg^–1.Scientific paper no. 8901-29, Department of Agronomy and Soils, College of Agriculture and Home Economics Research Center. Washington State University, Pullman, WA 99164, USA.Scientific paper no. 8901-29, Department of Agronomy and Soils, College of Agriculture and Home Economics Research Center. Washington State University, Pullman, WA 99164, USA.     

Free metal activity and total metal concentrations as indices of micronutrient availability to barley [Hordeum vulgare (L.) ‘Klages’]

The form in which a micronutrient is found in the rhizosphere affects its availability to plants. We compared the availability to barley of the free hydrated cation form of Fe³⁺, Cu²⁺, Zn²⁺, and Mn²⁺ versus their total metal concentrations (free ion plus complexes) in chelator-buffered solutions. Free metal ion activities were estimated using the chemical equilibrium program GEOCHEM-PC with the corrected database. In experiment 1, barley was grown in nutrient solutions with different Fe³⁺ activities using chelators to control Fe levels. Chlorosis occurred at Fe³⁺ activities of 10^–18 and 10^–19 M for barley grown in HEDTA and EDTA solutions, respectively. In experiment 2, barley was grown in nutrient solutions with the same calculated Fe³⁺ activity and the same chelator, but different total Fe concentrations. Leaf, root and shoot Fe concentrations were higher from CDTA buffered solutions which had the higher total Fe concentration indicating the importance of the total Fe concentration on Fe uptake. Results from treatments using EDTA or HEDTA, with one exception, were similar to the results from the CDTA treatment. This suggests differences in critical Fe³⁺ activities found in experiment 1 were due to differences in the total Fe concentration and not errors in chelate formation constants used to estimate the critical activities. Results for Cu, Zn, and Mn were similar to Fe; despite solutions with equal free Cu²⁺, Zn²⁺ and Mn²⁺ activities, plant concentrations of these metals were generally greater when grown in the solutions with the greater total amount of Cu, Zn, or Mn. When the free Zn²⁺ activity was kept constant while the total amount of Zn was increased from 4.4 to 49 M, leaf Zn concentration increased from 77 to 146 g g^-1. In order to predict metal availability to barley and other species in chelator-buffered nutrient solutions, both free and total metal concentrations in solution must be considered. The critical Fe³⁺ activities required by barley in this study are much higher than those from tomato and soybean, 10^-28 M, which strongly supports the Strategy 2 model of Fe uptake for Poaceae. This is related to the importance of the Fe³⁺ (barley) and the Fe²⁺ (tomato and soybean) ions in Fe uptake. Fe-stressed barley is known to release phytosiderophores which compete for Fe³⁺ in the nutrient solution, while tomato and soybean reduce Fe³⁺ to Fe²⁺ at the epidermal cell membranes to allow uptake of Fe²⁺ from Fe³⁺ chelates in solution.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetracetic acid - DTPA diethylenetriaminepentacetic acid - EDTA ethylenediaminetetracetic acid - EDDHA ethylenediamine-di(o-hydroxyphenylacetic acid) - HBED-N,N di(2-hydroxybenzoyl)-ethylenediamine-N,N-diacetic acid - HEDTA-N hydroxyethylenediaminetriacetic acid - MES-2 (N-morpholino)ethanesulfonic acid - NTA nitrilotriacetic acid     

Novel nutrient solutions for zinc nutrition research: buffering free zinc2+ with synthetic chelators and P with hydroxyapatite

Chelator-buffered nutrient solutions, in which computed free Zn²⁺ activities are buffered at 10^-10.0 M by including an excess of a synthetic chelator such as EDTA, have recently shown promise as a means of precisely regulating Zn nutritional status. A further refinement that would eliminate the confounding effect of high (and often phytotoxic) shoot P concentrations in solution-grown, Zn-deficient plants is also desirable. Several crop species were grown in 120-L of HEDTA-buffered solutions that contained just 10+-1 M P. Critical free Zn²⁺ activities ranged from 10 to 60 pM, and relative yields as low as 32% of control were achieved. Concentrations of P in the older leaves were very high (up to 46 mg g^-1) at low (Zn²⁺), suggesting that P toxicity can occur even without the high P concentrations (about 1 mM) typically used in Hoagland-type solutions. A second study was undertaken to better simulate soil conditions, wherein diffusion of P from the solid phase to the root is rate-limiting. Commercial hydroxyapatite (HAP) was enclosed in a pouch constructed of dialysis tubing, such that dissolution and diffusion occurred in response to plant depletion of P. Maize (Zea mays L.) and wheat (Triticum aestivum L.) can be supplied with P at adequate levels using this approach, and acutely Zn-deficient plants did not hyperaccumulate P. However, two dicots tested were too P-inefficient to grow normally with HAP as the sole P source.     

Bacterial sodium ion-coupled energetics

For many bacteria Na⁺ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na⁺ pumps in bacteria, the Na⁺-translocating oxaloacetate decarboxylase ofKlebsiella pneumoniae and the Na⁺-translocating F₁F₀ ATPase ofPropionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na⁺ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound -subunits including two conserved aspartates that may be important for Na⁺ translocation. The coupling ratio of the decarboxylase Na⁺ pumps depended on and decreased from two to zero Na⁺ uptake per decarboxylation event as increased from zero to the steady state level.InP. modestum, is generated in the course of succinate fermentation to propionate and CO₂. This is used by a unique Na⁺-translocating F₁F₀ ATPase for ATP synthesis. The enzyme is related to H⁺-translocating F₁F₀ ATPases. The F₀ part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of anEscherichia coli deletion mutant was completely functional as a Na⁺-ATP synthase conferring theE. coli strain the ability of Na⁺-dependent growth on succinate. The hybrid consisted of subunits a, c, b, and part of fromP. modestum and of the remaining subunits fromE. coli. Studies on Na⁺ translocation through the F₀ part of theP. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na⁺ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.     

Inhibition of Wheat Nitrate Reductase Activity by Zinc

The effect of Zn^2- on nitrate reductase (NR, EC 1.6.6.1) activity was studied in botá wheat (Triticum aestivum cv. Oasis) leaves and in the NR enzyme partially purified from wheat leaves. Leaf segments were floated on 0 to 5 mM ZnSO₄ solutions (pH 6.0) for 24 h under continuous light. Zn^2- at 250 M decreased NR activity and increased membrane permeability. However, parameters of cellular oxidative damage were scarcely affected by Zn^2- treatments. Accordingly, the decrease of NR activity induced by Zn^2- was not prevented by benzoate (a scavenger of oxygen radicals). The effect of Zn^2- was dependent on leaf age: it decreased NR activity in mature but not in young leaves. Zn² inhibited the partially purified NR. This inhibition was not reversed by either co- or post-incubation with cysteine, and the amount of -SH groups of the purified NR was not affected by Zn²⁺ indicating that Zn^2- inhibition does not involve key -SH groups of the enzyme. However, o-phenantroline both prevented and reversed Zn²⁺-induced NR inhibition. We concluded that the effect of Zn²⁺ on NR activity in vivo is not associated with an increase in active oxygen generation and involves a direct and reversible inhibition of the enzyme.