Effects of External KCl on Electrical Properties and K$(86Rb) Transport in the Elongating Region of Intact Phaseolus mungo Roots

Two simultaneous measurements, extracellular potential V andK^$(⁸⁶Rb) transport, and the intracellular potential of corticalcell E and potential V, were used to study the effects of externalKCl on two-day-old bean roots. High, external KCl concentrations(>10 mM) markedly enhanced K^$ loss from tissues in the elongatingregion to the external solution and induced depolarization ofthe membrane potential difference (PD=V–E). When Phaseolus roots were returned to a solution with a lowerconcentration of K^$, the K^$ loss and the potential difference,PD, were restored to their previous values. K^$ transport fromother parts of the root to the elongating region, however, didnot recover, and the potential, E, increased. These resultsclearly demonstrate that treatment of Phaseolus roots with ahigh external K^$ concentration inhibits K^$ translocation throughthe stele to the elongating cortical cells and is dependenton depolarization of the intracellular potential. (Received October 14, 1983; Accepted January 20, 1984)     

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M_r = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.     

The Effect of Cycloheximide on Absorption of Ions by Bean (Phaseolus vulgaris L) Roots

10^–7 M cycloheximide inhibited bean (Phaseolus vulgaris)root elongation by about 20 per cent but it inhibited absorptionof rubidium, sodium, and phosphate ions to a much greater extent(34–71 per cent). Tips of intact plant roots grown inthe inhibitor showed more inhibition in ion uptake than adjacentproximal portions of the same roots and this is taken to indicatethat 10^–7 M cycloheximide does not exert its effect onion uptake by any uncoupling action. Sodium uptake from 0.5 or 10 mM NaCl solutions by root tipswas inhibited by 10^–7 M cycloheximide to twice the extentthat it was in the elongating region of the root. Assuming thatthe inhibitor affects the plasmalemma more than the tonoplast,Epstein's model of parallel operation of system 1 and system2 at the plasmalemma is supported.     

Effects of Ca on Amino Acid Transport and Accumulation in Roots of Phaseolus vulgaris

Ca²⁺ stimulates the uptake of α-aminoisobutyric acid (AIB) into excised or intact Phaseolus vulgaris L. roots by a factor of two. In roots depleted of Ca²⁺ by preincubation with ethylenediaminetetraacetate, ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid, or streptomycin, the stimulatory effect is 7- to 10-fold. In the presence of Ca²⁺, roots accumulate AIB more than 100-fold; Ca²⁺-depleted roots only equilibrate with AIB. Radioautography shows [¹⁴C]AIB to be present in all cells after 90 min. Although Ca²⁺-depleted roots lose accumulated [¹⁴C]AIB about 10 times faster than roots supplied with Ca²⁺, this increased efflux is not the main cause for the decrease in net uptake observed. The latter is rather due to a less negative membrane potential Δψ in Ca²⁺ depleted roots (−120 mV → −50 mV). The basic feature explaining all the results of Ca²⁺ deficiency is an increase in general membrane permeability. No indication of a specific regulatory function of Ca²⁺ in membrane transport of roots has been obtained.     

Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.     

Amitrole Absorption by Bean (Phaseolus vulgaris L. cv ;Red Kidney') Roots : Mechanism of Absorption

The mechanism of transport of the herbicide 3-amino-1,2,4-triazole (amitrole) into Phaseolus vulgaris roots appears to be passive, as judged by the effect of temperature (Q₁₀ = 1.3 between 15 and 25°C) and the lack of sensitivity to metabolic inhibition afforded by 2,4-dinitrophenol and NaN₃. Amitrole absorption is a linear function of external concentration over several orders of magnitude and, thus, is not facilitated by a carrier mechanism. The absorption of amitrole is sensitive to external pH, being stimulated under acid conditions. This stimulation of amitrole absorption is seen at low (≤1 millimolar) amitrole concentrations, but not at high (50 millimolar) amitrole levels. While the apparent octanol-water partition coefficient varies with the pH of the aqueous phase, there is no clear correspondence between absorption and the apparent partition coefficient. Roots do not accumulate amitrole above concentration equilibrium; however, at a time when the net amitrole content of the root tissue begins to saturate, amitrole can be detected in the xylem stream. On a fresh-weight basis, amitrole absorption by roots is equal to that accomplished by trifoliate-leaf tissue. An estimate of the permeability coefficient (according to the analysis of Tyree et al. 1979 Plant Physiol 63: 367-374) suggests that amitrole possesses near-optimal permeability for an ambimobile solute, on the order of 2.12 (± 0.47) × 10⁻⁹ meters per second.     

Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.     

The Effects of Temperature on 86Rb Uptake by Two Species of Chlamydomonas (Chlorophyta, Chlorophyceae)

⁸⁶Rb uptake was examined in two species of unicellular greenalgae, Chlamydomonas nivalis isolated from snow, and a cellwall-less mutant of the temperate freshwater Chlamydomonas reinhardii.In C. reinhardii cells grown at 20°C and cooled rapidlyto 0°C, ⁸⁶Rb uptake was abolished. Cells cooled rapidlyto –5°C in the absence of ice accumulated ⁸⁶Rb veryrapidly but the time course of this uptake suggested non-selectiveaccumulation through a damaged plasmalemma. Cells grown at 8°Cwere viable, able to divide and motile; they showed no signsof cold-shock and ⁸⁶Rb uptake, albeit slow, was measurable at–5°C in the absence of extracellular ice. Cells ofC. nivalis grown at 20°C were damaged at sub-zero temperaturesalthough they did show an enhanced ⁸⁶Rb uptake at 0°C. Cellsgrown at 5°C were able to accumulate ⁸⁶Rb from media undercooledto -5°C in the absence of extracellular ice, and again showedenhanced uptake at 0°C. The process of acclimation to lowtemperature appears to differ in the two species. Key words: Chlamydomonas, temperature, ⁸⁶Rb uptake, membrane     

P-Protein Crystals do not Disperse in Uninjured Sieve Elements in Roots of Runner Bean (Phaseolus multiflorus) fixed with Glutaraldehyde

Light and electron microscopy of the P-protein (slime) crystalsin sieve elements in roots of the runner bean (Phaseolus multiporus)shows that the crystals occur and persist routinely in maturelater-formed sieve elements. Both components of the crystals,tails and central bodies, remain compact and undispersed. Thiscondition contrasts with that reported in stems in which previousauthors have shown, by means of electron micrographs, that thecentral bodies of crystals fixed in glutaraldehyde seem to disperseas the sieve elements differentiate. I suggest that stems maybe slowly enough fixed by glutaraldehyde for central bodiesto have time to respond to loss of turgor pressure but thatmore permeable roots may be more quickly fixed and may not facethe same circumstance.     

Effects of Angular Leaf Spot and Rust on Leaf Gas Exchange and Yield of Common Bean (Phaseolus Vulgaris)

Isolated and interactive effects of angular leaf spot (caused by Phaeoisariopsis griseola) and rust (caused by Uromyces appendiculatus) on leaf gas exchange and yield was studied in common bean (Phaseolus vulgaris L. cv. Carioca) plants. Gas exchange was measured on 37, 44, 51, and 58 d after planting using a portable photosynthesis system. The inoculation of plants with P. griseola (P), U. appendiculatus (U), and the combination of both pathogens (P+U) caused a significant reduction of net photosynthetic rate (P _N) and yield. The reduction of stomatal conductance (g _s), P _N, and yield was higher under P and combination of P+U than under U treatment. By effect of U, the reduction on yield was higher than the reductions on gas exchange parameters. On the treatment P+U, a reduction of 23 % in P _N and a correspondent reduction of 32 % in yield was observed. The interactive effects of the pathogens on yield could be explained in part by the decreases in g _s and in P _N of diseased bean leaves. The combined effect of both diseases on yield and gas exchange parameters suggests an antagonistic interaction.     

耐低钾籼稻幼苗根部的K~ (~(86)Rb~ )运输和通量分析

用通量分析的方法比较研究籼稻73—07和税稻80—66根部K~ (~(86)Rb~ )的吸收和运输的差异。耐低钾的籼稻73—07根部吸收和运输K~ (~(86)Rb~ )的速率显著高于不耐低钾的釉稻80—66。前者的J_(oc)和J_(cx)分别为后者的5.9和5.6倍。籼稻73—07根部的Q_c和Q_v均高于籼稻80—66。由于上运速度快,籼稻73—07的t_c/2显著小于籼稻80—66,表明籼稻73—07根部的K~ (~(86)Rb~ )周转快,有利于根部从低钾介质中吸收K~ 。     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Influence of Light on the Bioelectric Potential of the Bean (Phaseolus vulgaris) Hypocotyl Hook

By use of surface electrodes electropotenlial measurements were carried out on hypocotyl hooks of Phaseolus vulgaris seedlings. The hooks were illuminated with a small spot of white, blue, red or far red light. The potential changes in bean hypocotyl hooks do not show the red-far red reversible characteristics of phytochrome-mediated processes. By experimenting with inhibitors of photosynthesis we could demonstrate that the light-triggered potential changes in green bean hooks are correlated to photosynthetic electron transport phenomena. The red-light-induced transient is a depolarization, whereas blue light induces a hyperpolarization. Etiolated beans exhibit no bioelectric potential changes when subjected to red or far red irradiations. Blue light and white light induce a strong hyperpolarization in etiolated hooks cells. This transient seems to be an action potential induced by light. The action potential is influenced by inhibitors of electron transport and oxidative phosphorylation. By comparing the action spectrum of the action potential induced by light with the absorption spectra of extracted carotenoids and xanthophylls from etiolated bean hypocotyl hooks, we observed similarities.     

Purification and Characterization of the Main Isozyme of Polyphenol Oxidase in Mung Bean (Vigna mungo) Seedlings

The induction of NADPH-generating enzymes by polychlorinated biphenyls (PCB) in rats was investigated. The administration of PCB to rats for 3 and 14 days increased the activities of malic enzyme (ME, EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44) about 2-fold above the control level in the liver. Hepatic mRNA levels of ME, G6PD, and 6PGD, except for G6PD mRNA of the 14-day group, were also elevated to the same degree as the enzyme activities in PCB-treated rats. In rats fed a PCB-containing diet for 1 day, the hepatic mRNA levels of ME and G6PD were elevated prior to the induction of enzyme activity. In the kidney, lung, spleen, heart, and testis, the mRNA levels of ME, G6PD, and 6PGD were not affected by PCB. The induction of hepatic NADPH-generating enzymes would imply an increased demand of NADPH in the liver of rats fed with a PCB-containing diet.     

Effects of Periodic- and Transient-Osmotic Stress on Electric Potentials in Bean Roots

Intact bean (Phaseolus mungo) roots were subjected to periodic-and transient-osmotic stress by treatment with a solution oflow water potential °. Both the intracellular potential,E, and the extracellular potential, V, were measured with amultimicrochamber system that combined intracellular microelectrodesand external electrodes. In elongating regions, ° simultaneouslyaffected potentials E and V. The response of E to ° couldbe presented by a transfer function that included dead timeand first order elements. These elements were consistent withthe emergence of an electromotive force, produced by respiration,between the cortex and the stele in the elongating region. (Received January 14, 1983; Accepted June 23, 1983)     

Response to Phosphate Deficiency in Bean (Phaseolus vulgarisL.) Roots. Respiratory Metabolism, Sugar Localization and Changes in Ultrastructure of Bean Root Cells

Bean plants (Phaseolus vulgarisL. ‘Zlota Saxa’)were cultured on complete (+P) or phosphate-deficient (-P) nutrientmedium. A large increase in glucose concentration was foundin the meristematic zone of -P roots compared to control roots.The increased glucose concentration in the meristematic zonedid not influence total respiration rate. Glucose or uncoupler(carbonyl cyanide m-chlorophenylhydrazone) failed to increasethe respiration rate in -P root segments, but stimulated respirationin +P roots. The ultrastructure of cortical cells from the meristematicroot zone showed marked differences between +P and -P plants.Large vacuoles, invaginations of the plasmalemma and condensedforms of mitochondria were dominating features in cortical cellsof -P roots. Analysis of extracts after treating roots withdimethylsulfoxide (DMSO) indicated different localization ofsugars in the cell compartments. In roots of -P plants, mostof the reducing sugars were detected in the cytoplasm fractionwhile most sucrose was in the vacuole. Observations of the effectof 10% DMSO on cell ultrastructure indicated partial destructionof the plasmalemma but not the tonoplast. The localization ofreducing sugars in secondary vacuoles or plasmalemma invaginationsin the cells from the meristematic region of -P roots is discussed.Copyright1998 Annals of Botany Company. Bean (Phaseolus vulgarisL.), roots, Pi deficiency, respiration, meristematic zone, ultrastructure, sugar efflux, reducing sugars and sucrose localization.     

Properties of (Sodium Potassium)-Stimulated ATPases in English Ryegrass Roots and Implications in Ion Transport

Maximum ATPase activities in the cell wall fraction of English ryegrass (Lolium perenne L.) roots were stimulated by foru discrete millimole ratios of (Na⁺+ K⁺); 40:0, 35:5, 5:35, and 0:40. The optimal pH for stimlation was found to be 6.5. Contrary to data in the literature, Mg²⁺ inhibited all stimulatory ratios of (Na⁺+ K⁺) when plants were cultured on an adequate nutrient solution. When grown on a dilute solution, Mg²⁺ enhanced (Na⁺+ K⁺)-stimulated ATPase activity in this membrane preparation. The single optimal combined concentration of (Na⁺+ K⁺) for all stimulatory ratios was 40 MM. The ratios of (Na⁺+ K⁺) which stimulated ATPase activity in the cell wall fraction varied with position along the root axis such that all rarely existed simultaneously nor did any exist in the terminal millimetre of the root. Both cell wall and microsomal fractions showed stimulation by (Na⁺+ K⁺) at all the above ratios indicating the possible presence of plasma membrane fragments in both fractions. Only the 35:5 ratio was stimulations were found in the supernatant. Implications of ion-stimulated ATPase involvement in ion transport were drawn from the appearance of ATPase activity at a 40:0 ratio of (Na⁺+ K⁺) and the disappearance of stimulations at 35:5, 5:35, and 0:40 ratios when plants were moved from a strong (35 mM total concentration) to a dilute (0.75 mM) nutrient solution.     

Transport and redistribution of selenium in the bean plant (Phaseolus vulgaris)

After incubation for 3 h with (⁷⁵Se) selenate, the selenium distribution in the bean plant (Phaseolus vulgaris L. cv. Contender) through a 29-day period showed an uneven distribution: roots and trifoliate leaves were richer in ⁷⁵Se than stem and primary leaves. The high selenium concentration of roots resulted from the retention of selenate by the root cells: at the end of the 29-day period about 60° of the radioactivity was always ethanol-soluble, and when analysed by paper chromatography, proved to be selenate. By contrast, much of the radioactivity of the leaves was ethanol-insoluble, ⁷⁵Se being quickly captured in metabolic processes which immobilize it. During plant development, a portion of the total selenium remains mobile and is continually mobilized to the younger organs which display a rapid growth rate. This delivery results from a progressive liberation of selenate retained by mature organs, especially the roots, and from turnover in older leaf tissues, especially the trifoliate leaves.     

Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.