Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Regulation of k influx in barley : effects of low temperature

The proteinases present in dark-germinated flax seeds (Linum usitatissimum) were studied as a function of germination at 25°C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45°C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedlings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved to be very reproducible and afforded detection and good quantification of various proteinase zones. An ethylenediaminetetraacetate-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). Ethylenediaminetetraacetate also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose-dependent manner. The addition of leupeptin, α-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate, or 1,10-phenanthroline prior to, during, or after exchange of Triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings.     

Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate

Barley plants were grown in nutrient solutions, which were maintained at either 0 (-P) or 15 μ M orthophosphate (+P). After 11 days phosphate influx into the intact roots of the -P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the -P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)^-1h^-1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r²= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of -P plants was rapidly reduced by the provision of 15 μ M orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)^-1 h^-1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)^-1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.     

Effect of root perturbation and excision on nitrate influx and efflux in barley (Hordeum vulgare) seedlings

The effects of perturbation and excision on net NO^-₃, uptake, influx and efflux in roots of 8-day-old barley ( Hordeum vulgare L.) seedlings induced with NO^-₃ or NO^-₂ were determined. Perturbation was simulated by mechanically striking the intact roots with a glass rod. Perturbation or excision of roots and subsequent division into small segments had little effect on NO^-₃ influx, but briefly inhibited net uptake which recovered within a few min. While in perturbed roots net uptake rates recovered to the same level as in control roots, full recovery did not occur in excised roots. Inhibition of net uptake was due to stimulation of NO^-₃ efflux. The recovery time and level of inhibition of net NO^-₃ uptake and/or stimulation of efflux were a function of extent of perturbation, or the number of segments following excision, and root NO^-₃ concentration. NO^-₃ efflux was further stimulated when roots were perturbed after cytoplasmic NO^-₃ had been depleted, indicating that both the plasmalemma and tonoplast may be affected. In excised roots both NO^-₃ influx and efflux decreased with age due to depletion of energy sources. The results indicate that root perturbation and excision had no effect on NO^-₃ influx but inhibited net uptake by stimulating efflux.     

Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx

The cytoplasmic NO₃⁻ concentration ([NO₃⁻]_c) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO₃⁻ concentration ([NO₃⁻]_o), [NO₃⁻]_c was estimated to be 0.66 mm for plants previously grown in 100 μm NO₃⁻. It increased linearly with [NO₃⁻]_o between 2 and 20 mm, up to 3.9 mm at 20 mm [NO₃⁻]_o. The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO₃⁻]_c for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO₃⁻]_c and cell membrane potentials of −200 to −300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO₃⁻ is possible via the high-concentration, low-affinity transport system for NO₃⁻. This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO₃⁻ against its electrochemical potential gradient.     

Effect of ATPase inhibitors on cell potential and k influx in corn roots

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Ψ) and K⁺ (⁸⁶Rb) influx of corn root tissue over a wide range of K⁺ activity. N,N′Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K⁺ influx and depolarized Ψ at low, but not at high, K⁺ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K⁺ influx over a wide range of K°, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Ψ_p) above 0.2 millimolar K°. Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K⁺ influx and Ψ_p at low K° can be attributed to inhibition of the plasmalemma ATPase.     

Potassium influx into maize root systems : influence of root potassium concentration and ambient ammonium

Potassium influx into roots of dark-grown decapitated maize seedling (Zea mays L., cv Pioneer 3369A) was examined in presence and absence of ambient ammonium and at various root potassium concentrations. Six-day old seedlings which were dependent on the endosperm reserves for their energy source were exposed to KCl (labeled with ⁸⁶Rb) ranging from 5 to 200 micromolar. At both low (13 micromoles per gram fresh weight) and high (100 micromoles per gram fresh weight) root potassium concentration, isotherms indicated two potassium influx systems, one approaching saturation at 50 to 100 micromolar potassium and an additional one tentatively considered to be linear. A mixed-type inhibition by ammonium for the low-concentration saturable system was indicated by a concomitant decrease in V_max and increase in K_m. High root potassium concentration decreased V_max but had little effect on K_m of this system. The rate constant for the second quasilinear system was decreased by ambient ammonium and by high root potassium status. Transfer of high potassium roots to potassium-free solutions resulted in an increase in influx within 2 hours; by 24 hours influx significantly exceeded that of roots not previously exposed to potassium. In roots of both low and high root potassium concentrations, potassium influx was restricted progressively as ambient ammonium increased to about 100 micromolar, but there was little further inhibition as ammonium concentrations increased beyond that to 500 micromolar. The data imply that potassium influx has two components, one subject to inhibition by ambient ammonium and one relatively resistant.     

An anomaly in potassium accumulation by barley roots: I. Effect of anions, sodium concentration, and length of absorption period

Excised barley roots accumulated 40 to 50% more K(+) from 0.04 mm than from 0.06 mm KCl when incubated for 24 hours in KCl solutions containing 0.2 mm CaSO(4). This phenomenon was not markedly influenced by the rate of absorption of the counteranion. The presence of Na(+) in the treatment solutions decreased total K accumulation but did not alter the K(+) concentration at which the accumulation peak occurred. Short interval studies indicated that this phenomenon is easily observable after 4 hours and begins to become apparent within 2 hours. In comparison with barley, accumulation of K(+) by excised wheat roots decreased as KCl concentration was increased from 0.02 to 0.06 mm; but K(+) accumulation curve for corn roots showed no peaks or depressions in the concentration range of 0.01 to 0.1 mm. A normal hyperbolic curve was noted for the accumulation of Na(+) from 0.01 to 1 mm NaCl by barley roots.     

Boron uptake by excised barley roots: I. Uptake into the free space

At 2 C, all boron accumulated by excised barley roots (Hordeum vulgare L. cv. Herta) remains in the free space; i.e. active uptake is nil at this temperature. Three component fractions of free space B were apparent: (a) a surface contaminant film of B on blotted roots, (b) water free space B, and (c) B reversibly bound in the cell walls. A stoichiometric release of H⁺ from the roots in the presence of B indicated that B was bound by borate complexes with polysaccharides in the cell walls. Polysaccharide-borate complexes are much less stable than those of monosaccharides, and the bound B fraction could be readily removed by rinsing the roots in the presence of a monomeric polyol possessing the necessary cis-diol configuration. Cell wall material separated from excised barley roots had a B binding capacity 66% greater than that of intact roots.     

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

The influence of monovalent cations upon influx and efflux of Ca2+ in barley roots

The effects of monovalent cations, inhibitors of metabolism dinitrophenol (DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and KCN and temperature variations upon Ca²⁺ fluxes in intact roots of barley (Hordeum vulgare L. cv. Fergus and Herta) seedlings were investigated. ⁴⁵Ca²⁺ influx was depressed in CaSO₄-grown (low-salt) plants by the presence of NH₄⁺, K⁺, or Na⁺ in the uptake medium. In contrast Ca²⁺ influx was slightly increased by Li⁺. In low-salt roots pretreated with KCN and in roots preloaded with K⁺ (high-K⁺ plants), the presence of K⁺ in the medium had no significant effect on Ca²⁺ influx, while in roots preloaded with Na⁺, the presence of K⁺ in the medium depressed Ca²⁺ influx. In absolute terms, Ca²⁺ influx was significantly greater in high-salt (both K⁺ or Na⁺ preloaded) than in low-salt roots.Patterns of ⁴⁵Ca²⁺ efflux in the absence and in the presence of K⁺, NH₄⁺, or Li⁺ in the external medium showed that these monovalent cations caused stimulation of ⁴⁵Ca²⁺ efflux both from the cytoplasmic and vacuolar phases.It was noted that these modifications of Ca²⁺ fluxes by monovalent cations are transient and characteristic of a transitional stage of cation uptake by low-salt roots. We conclude that, together with stimulated active H⁺ efflux (another characteristic of this transitional stage), modifications of Ca²⁺ fluxes during monovalent cation uptake by low-salt roots is a response directed towards the maintenance of electrical neutrality.Determination of net fluxes revealed that the plants were close to Ca²⁺ flux equilibrium in the growth medium (0.5 mM CaSO₄). Transfer of these plants to 0.5 mM CaSO₄ + 0.25 mM K₂SO₄ caused a net release of CA²⁺ into the external medium.     

Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore.

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = approximately 265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30-100 microM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

Longitudinal variation in cadmium influx in sunflower (Helianthus annuus L.) roots as depending on the growth substrate,root age and root order

Aims

In a previous work, we observed a longitudinal decrease in Cd²⁺ influx starting from the root tip in first order lateral roots of sunflower (Helianthus annuus L.) grown in hydroponics. This variable influx was expected to impact the total Cd²⁺ uptake depending on the root system architecture and on how steep was the decrease of the influx. Here, we examined the influence of the culture substrate, of the age and order of lateral roots on the longitudinal variation of Cd²⁺ influx.

Methods

By using short-term exposures to ¹⁰⁹Cd-labelled solution (5 to 200 nM), we compared the longitudinal variations in Cd²⁺ roots influx depending on the growth substrate (hydroponics or sand), on the root age and order.

Results

In second order laterals, Cd²⁺ influx decreased from the apex to the root base, as for first order laterals. For sand cultures compared to hydroponics, the mean Cd²⁺ influx was lower and decreased more steeply with the distance from the apex. The influx also decreased with increasing root age and order, markedly in hydroponics but less for sand cultures.

Conclusion

Results suggested that for a given root surface area, the Cd²⁺ uptake by a root system should increase with increasing number of root tips and decreasing individual root length.     

Studies on the mucilaginous layer of barley (Hordeum vulgare) roots

The nature and composition of the external, mucilageneous layer of barley roots was studied by extraction methods and electron microscopy. Barley roots were extracted with chloramphenicol-supplemented water at 35°C, with NH₄Cl at various concentrations and with pectinase solutions. The kinetics of transfer of bacteria, total and reducing sugars, proteins, Ca⁺⁺ and K⁺ was studied, and the removal of the mucigel from the extracted roots was followed under the electron microscope. Within 2 to 3 hours of treatment with water, the rate of release of sugars, ions, proteinaceous material and bacteria, was reduced to almost zero. Increasing concentrations of ammonium chloride enhanced transfer of ions to the extracting solution but affected sugar extraction to a lesser extent. Electron micrographs of ammonium chloride-extracted roots revealed that the amorphous, rather than the fibrillar fraction of the mucigel was removed. At 10³ meq of NH₄Cl, distortion of the epidermal layer of the extracted roots was observed. With pectinase as an extractant, there was some enhancement of sugars and ions transfer from the roots to the extracting solution. Electron micrographs showed that the main site of extraction of pectinase was the boundary layer between the root surface and the mucigel. Paper chromatography of the acid hydrolyzate of the water extracted, ethanol-precipitated fraction showed the presence of compounds identical in Rf values to D-glucose, D-arabinose, D-glucuronic acid and D-galacturonic acid. Present methods available for the extraction of the mucigel do not allow the differentiation between extracted pectic compounds which originate from the internal root tissue, and the mucigel. re]19751128 Dept. of Field Crops Dept. of Soil and Water Sciences Dept. of Microbiology and Phytopathology     

Phosphorus (P) uptake and growth of a root hairless barley mutant (bald root barley, brb) and wild type in low- and high-P soils

The recently isolated root‐hairless mutant of barley (Hordeum vulgare L), bald root barley, brb offers a unique possibility to quantify the importance of root hairs in phosphorus (P) uptake from soil. In the present study the ability of brb and the wild‐type, barley genotype Pallas producing normal root hairs to deplete P in the rhizosphere soil was investigated and the theory of diffusion and mass flow applied to compare the predicted and measured depletion profiles of diffusible P. Pallas depleted twice as much P from the rhizosphere soil as brb. The P depletion profile of Pallas uniformly extended to 0.8 mm from the root surface, which was equal to the root hair length (RHL). The model based on the theory of diffusion and mass flow explained the observed P‐depletion profile of brb, and the P depletion outside the root‐hair zone of Pallas, suggesting that the model is valid only for P movement in rhizosphere soil outside the root‐hair zone. In low‐P soil (P in soil solution 3 µm ) brb did not survive after 30 d, whereas Pallas continued to grow, confirming the importance of root hairs in plant growth in a P‐limiting environment. In high‐P soil (P in soil solution 10 µm ) both brb and Pallas maintained their growth, and they were able to produce seeds. At the high‐P concentration, RHL of the Pallas was reduced from 0.80 ± 0.2 to 0.68 ± 0.14 mm. In low‐P soil, P‐uptake rate into the roots of Pallas was 4.0 × 10^?7 g mm^?1 d^?1 and that of brb was 1.9 × 10^?7 g mm^?1 d^?1, which agreed well with the double amount of P depleted from the rhizosphere soil of Pallas in comparison with that of brb. In high‐P soil, the P uptake rates into the roots of brb and Pallas were 3.3 and 5.5 × 10^?7 g mm^?1 d^?1, respectively. The results unequivocally confirmed that in a low‐P environment, root hairs are of immense importance in P acquisition and plants survival, but under high‐P conditions they may be dispensable. The characterization of phenotypes brb and Pallas and the ability to reproduce seeds offers a unique possibility of molecular mapping of QTLs and candidate genes conferring root‐hair formation and growth of barley.