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1.
The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.  相似文献   

2.
A pencil-type antimony microelectrode of double-barreled design with a tip of less than 1 to 2 μm in outside diameter was constructed and used to measure intracellular pH(pHi) on frog sartorius muscle and renal tubular cells. Simultaneous observations of membrane potential difference (EM) were made. The results obtained were as follows: (1) The in vivo pHi of frog sartorius muscle was 7.12 ± 0.07 (SD) (n = 144); the simultaneously measured EM was -51.1 ± 7.9 mV. The in vivo pHi of frog proximal tubule was 7.49 ± 0.07 (n = 221) and the EM peri across the peritubular membrane was -50.2 ± 9.0 mV. (2) In proximal tubule in vivo, there was a negative correlation between pHi and EM (r = -.62, p <. 05). On the other hand, in sartorius muscle in vivo, a positive correlation between the two was found (r =. 85, p <. 001). (3) In in vitro sartorius muscle, the pHi was 7.03 ± 0.14 (n = 9) and EM was -62.4 ± 4.4 mV within one hour after isolation. (4) Increasing the external potassium concentration in the preparations to 75 mM caused a progressive depolarization by 43.3 ± 15.9 (m = 4) mV, while pHi changed in the alkaline direction by 0.22 ± 0.03 pH unit. (5) These results indicate that the pHi in both tissues does not obey the Donnan rule.  相似文献   

3.
Accumulation of sodium and rubidium by excised barley roots was investigated. The concentration isotherm yielded one absorption shoulder. Nevertheless, it is suggested that two mechanisms take part in the uptake of sodium and rubidium: One non-metabolic mechanism with an apparent participation at low external salt concentrations (< 1 mM) and at high concentrations (> 20 mM). Such a mechanism is almost unaffected by low temperature conditions and by metabolic inhibitors. Rubidium possesses a high affinity toward this non-metabolic system. The second mechanism is sensitive to metabolic inhibitors and to low temperature conditions. It dominates at intermediate external concentrations (1–20 mM). Sodium possesses high affinity towards this mechanism. The two mechanisms operate in a parallel manner beyond a diffusion barrier (= plasmamembrane) surrounding the cells. It is assumed that both the metabolic and the non-metabolic mechanisms operate in the entire concentration spectrum, but their relative contribution to the total uptake varies at different ranges.  相似文献   

4.
Cell-wall preparations from barley roots contain about 20 percent of the ATP-ase activity of the whole homogenate. This activityis maximal near pH 7, activated by calcium and magnesium ionsand shows further activation when sodium and potassium chloridesare applied at alkaline pH values. High concentrations of sodiumchloride and ammonium sulphate are needed to elute the activityfrom the walls which suggests an ionic binding with the wallfraction. Excised root tips release inorganic phosphate fromATP with no lag phase, and this activity shows a response tovariation in substrate and magnesium concentration similar tothat of the cell-wall preparations, suggesting a surface locationof the enzyme. The properties of this hydrolytic activity arediscussed in relation to those described in other plant systemsand to animal transport ATP-ases.  相似文献   

5.
6.
Determination of Nitrate Reductase Activity in Barley Leaves and Roots   总被引:4,自引:0,他引:4  
The inactivation of nitrate reductase in the leaves and rootsof barley (Hordeum vulgare L. cv. Mazurka) during and afterextracting was investigated. At 0 °C in the absence of casein,25 per cent of ‘total’. i.e. maximal in vitro, nitratereductase activity was lost during the 2 min extraction process,followed by a slower loss of activity while the extract wasstored in ice. Activity was maintained by adding a minimum of1 per cent casein to the extraction medium containing 0·1M phosphate (pH 7·5), 1 mM EDTA and 1 mM dithiothreitol.Nitrate reductase was stable for several hours in these extracts,but declined in a first order manner in the absence of dithiothreitol.Casein also prevented the initial loss while making root extracts,but had less effect during storage. Using casein and thiols, nitrate reductase activity in light,(as product of maximal in vitro rates and wt g–1) in leaveswas 98 per cent of the total activity in 31-day-old plants grownwith full nutrient in water culture and 60-day-old field-grownplants receiving no fertilizer. Field-grown plants, however,exhibited only 17 per cent of the activity of culture-grownplants. Nitrate reductase in leaves of barley plants grown in waterculture had a diurnal rhythm. During the first 3 h of the lightperiod, activity increased to 1·3 x the ‘dark’value. This was followed by a temporary decrease and then byanother increase to a maximum of 1·7 x the ‘dark’value, occurring about 8 h after illumination. Activity thendecreased during the rest of the light period and in darkness. Hordeum vulgare L., barley, nitrate reductase  相似文献   

7.
8.
The total uptake and relative distribution of Na+ along the root was investigated in excised barley roots (Hordeum vulgare L. cv. Union) of 6-day-old plants cultured on 0.25 mM CaSO4. One technique involved cutting the roots at harvest time after different uptake periods into different segments each 2 cm long. These segments were ashed and sodium was measured by flame photometry. For each experiment two treatments were done, one containing calcium in the uptake solution and one without calcium. A second technique involved the measurement of sodium transport, using labelled 22Na+ solution. Although no significant differences could be found between the calcium containing and the calciumless treatments at 24 h, there were clearcut differences for the first 6 to 8 h. The effect of calcium on the first stages of the sodium uptake was a large accumulation of sodium in the part close to the apex with a translocation close to nil; whereas the sodium taken up in the absence of calcium did not accumulate in such large amounts in the apex region but was immediately transported basipetally.  相似文献   

9.
Nitrate reductase (NO3R) activity, nitrite reductase (NO2R) activity and NADH2 dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10?5; 3 × 10?5; 5 × 10?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1?1, that is 4.65 × 10?7;4.65 × 10?6 and 2.3 × 10?5 M). NO3R activity was not influenced by IAA, NO2R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO3R activity significantly both after 9 h and 24 h incubation, slightly increased NO2R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO2R activity. Nevertheless the depressing effect of kinetin on NO3R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.  相似文献   

10.
The short-lived radiotracer 13N was used to study feedback regulation of nitrate influx through the inducible high-affinity transport system of barley (Hordeum vulgare L. cv Steptoe) roots. Both wild-type plants and the mutant line Az12:Az70 (genotype nar1a;nar7w), which is deficient in the NADH-specific and NAD(P)H-bispecific nitrate reductases (R.L. Warner, R.C. Huffaker [1989] Plant Physiol 91: 947-953) showed strong feedback inhibition of nitrate influx within approximately 5 d of exposure to 100 fmu]M nitrate. The result with the mutant, in which the flux of nitrogen into reduced products is greatly reduced, indicated that nitrate itself was capable of exercising feedback regulation upon its own influx. This conclusion was supported by the observation that feedback in wild-type plants occurred in both the presence and absence of L-methionine sulfoximine, an inhibitor of ammonium assimilation. Nitrite and ammonium were also found to be capable of exerting feedback inhibition upon nitrate influx, although it was not determined whether these ions themselves or subsequent metabolites were responsible for the effect. It is suggested that feed-back regulation of nitrate influx is potentially mediated through several nitrogen pools, including that of nitrate itself.  相似文献   

11.
Previous papers have shown that abscisic acid can inhibit transportof ions across the root to the xylem vessels, resulting in reducedexudation from excised roots or inhibiting guttation from intactplants. However, it has not been established whether the inhibitionwas due to a reduction in salt transport (Js) or in permeabilityof the roots to water (Lp). This paper investigates the effectof ABA on Lp and Js separately. It is shown that Lp increasedin ABA and then fell, but was about the same as in control rootswhen transport was inhibited. The effect of ABA on exudationtherefore appeared to be mainly due to reduction in Js. Inhibitionof Js was also present in intact, transpiring plants and sowas not due to reduced water flow. The inhibition of ion releaseto the xylem affected Na+, Mg2+, Ca2+, and phosphate as wellas the major ion in the exudate, K+. It is concluded that ABAinhibits salt transport to the shoot by acting on ion transportinto the xylem, and not by reducing water flow coupled withsalt transport.  相似文献   

12.
13.
14.
We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO3 and H+ within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO3 uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H+-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO3 (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO3 absorption. These results strongly indicate that the NO3 electrical response is due to the operation of a NO3 transport system in the plasmalemma of maize root cells. Furthermore, the results from the H+-ATPase inhibitor studies indicate that the NO3 transport system is linked to the H+-ATPase, presumably as a NO3/H+ symport. This is further supported by the pH response of the NO3 transport system (inhibition at alkaline pH values) and the change in net H+ flux from a moderate efflux in the absence of NO3, to zero net H+ flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO3/H+ flux data is a NO3/H+ symport with a NO3:H+ flux stoichiometry >1, whose operation results in the stimulation of the H+-ATPase due to the influx of protons through the cotransport system.  相似文献   

15.
The effects of carbon dioxide concentrations up to 8 per centin air on uptake of potassium and chloride at two pH levels,nominally pH 6 and 8, werestudied. In all experiments, enhanced uptake of potassium occurred atthe higher pH level with carbon dioxidefree air, but chlorideuptake was generally unaffected. At nominal pH 6, 1 per cent carbon dioxide reduced and 6 percent increased potassium uptake. There was no effect on chlorideuptake except with 1 per cent carbon dioxide where a markedenhancement was recorded. At nominal pH 8, l and 2 per cent carbon dioxide increased potassiumuptake whereas 6 and 8 per cent were inhibitory. Chloride uptakewas favoured by 1 and 8 per cent concentrations of the gas.  相似文献   

16.
Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)  相似文献   

17.
Net fluxes of NH4+ and NO3 into roots of 7-day-old barley (Hordeum vulgare L. cv Prato) seedlings varied both with position along the root axis and with time. These variations were not consistent between replicate plants; different roots showed unique temporal and spatial patterns of uptake. Axial scans of NH4+ and NO3 net fluxes were conducted along the apical 7 centimeters of seminal roots of intact barley seedlings in solution culture using ion-selective microelectrodes in the unstirred layer immediately external to the root surface. Theoretically derived relationships between uptake and concentration gradients, combined with experimental observations of the conditions existing in our experimental system, permitted evaluation of the contribution of bulk water flow to ion movement in the unstirred layer, as well as a measure of the spatial resolution of the microelectrode flux estimation technique. Finally, a method was adopted to assess the accuracy of this technique.  相似文献   

18.
Aslam M  Travis RL  Rains DW 《Plant physiology》1996,112(3):1167-1175
Induction of an NO3- efflux system in intact barley (Hordeum vulgare L.) roots was demonstrated. Since the measurement of NO3- efflux is dependent on its accumulation, experiments were devised to facilitate accumulation under noninducing conditions. This was accomplished by incubating seedlings in 10 mM NO3- in the presence of RNA and protein synthesis inhibitors. Under these conditions NO3- uptake is mediated by constitutive high- and low-affinity transport systems. Control roots were incubated with 1.0 mM NO3-. This resulted in the accumulation of similar levels of NO3- in both treated and control roots; however, cytoplasmic NO3- efflux from inhibitor-treated roots was much lower than from control roots. Following a brief lag period, efflux rates increased rapidly in the presence of NO3- for 8 to 12 h. The NO3- efflux system was also induced by ambient NO2-. After induction the efflux system was relatively stable in the presence of RNA and protein synthesis inhibitors as long as NO3- or NO2- was present. These results suggest that NO3- efflux may be an inducible system requiring both RNA and protein synthesis, as does induction of the uptake system. The efflux system, however, has a much slower turnover rate than the uptake system.  相似文献   

19.
The unidirectional Ca2+ fluxes across the plasma membrane andtonoplast were determined in both excised roots and roots ofintact seedlings of rye (Secale cereale L. cv. Rheidol). Theunidirectional Ca2+ fluxes across the plasma membrane and tonoplastmeasured in excised roots were of a similar order of magnitudeto those determined in roots of intact plants. Influx and effluxof Ca2+ across the root plasma membrane were similar (estimatedto be between 0·7 and 3·4 µmol g  相似文献   

20.
A simple technique to grow excised tomato roots in sand in sterileculture is described. The apparatus consists of a culture tubefilled with sand linked to a small flask containing the nutrientsolution. By raising the lower-placed flask the sand is flushedwith solution, thereby replenishing nutrients at the root surface.Lowering the flask again allows air to enter the sand, thuseffecting sufficient aeration. It is further demonstrated that growth of the roots is relatedto acration and not to mechanical impedance in sands of differentgrain size.  相似文献   

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