The calcium nutrition of bambara groundnut (Vigna subterranea (L.) Verdc.)

Iron chlorosis induced by Fe-deficiency is a widespread nutritional disorder in many woody plants and in particular in grapevine. This phenomenon results from different environmental, nutritional and varietal factors. Strategy I plants respond to Fe-deficiency by inducing physiological and biochemical modifications in order to increase Fe uptake. Among these, acidification of the rhizosphere, membrane redox activities and synthesis of organic acids are greatly enhanced during Fe-deficiency. Grapevine is a strategy I plant but the knowledge on the physiological and biochemical responses to this iron stress deficiency in this plant is still very poor. In this work four different genotypes of grapevine were assayed for these parameters. It was found that there is a good correlation between genotypes which are known to be chlorosis-resistant and increase in both rhizosphere acidification and Fe^III reductase activity. In particular, when grown in the absence of iron, Vitis berlandieri and Vitis vinifera cv Cabernet sauvignon and cv Pinot blanc show a higher capacity to acidify the culture medium (pH was decreased by 2 units), a higher concentration of organic acids, a higher resting transmembrane electrical potential and a greater capacity to reduce Fe^III-chelates. On the contrary, Vitis riparia, well known for its susceptibility to iron chlorosis, fails to decrease the pH of the medium and shows a lower concentration in organic acids, lower capacity to reduce Fe^III and no difference in the resting transmembrane electrical potential. H Marschner Section editor     

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = −220 millivolts). In plants maintaining a low pd (−110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H⁺ cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H⁺ -extrusion pump.     

Proton efflux and transfer cell formation as responses to Fe deficiency of soybean in nutrient solution culture

Hydroponically grown Hawkeye soybeans with N supplied as NO ₃ ^– did not show any measurable pH decrease of the nutrient solution during the first week of Fe deficiency as has been observed for other Fe-efficient dicotyledonous species. Only after prolonged Fe stress with no renewal of the nutrient solution could an unspecific pH reduction be measured as a consequence of a decrease in the NO ₃ ^– content of the solution. On the other hand, Fe stress induced H⁺ efflux could be localized at the root tip region by day foru of-Fe treatment when intact plants were transferred from the nutrient solution to agar medium containing the pH indicator dye bromocresol purple. However, the activity of this H⁺ pump obviously was too weak to neutralize HCO₃-ions simultaneously excreted from older root parts and to acidify the bulk nutrient solution. Thus no remobilization of iron precipitated on older parts of the roots occurred and the plants remained chlorotic.Electron microscopy of the H⁺ extruding zone revealed hypodermal transfer cells with wall protuberances surrounded by cytoplasm especially rich in mitochondria. No transfer cells occurred in the rhizodermis as seen in other Fe-efficient dicots. Some cortical cells also showed transfer cell features with wall protuberances in the intercellular spaces. Often wall ingrowths were surrounded by a periplasmic space which reduced the potential surface amplification of the plasma membrane. It is concluded that the weak capacity of Hawkeye soybeans for Fe stress-induced H⁺ extrusion correlates with their less intense wall labyrinth formation as compared with other dicotyledonous species with higher Fe efficiency.     

Electrical noise measurements on red beet vacuoles : another way to detect the ATPase activity

The vacuolar potential (V_vac) and its fluctuations were recorded in red beet vacuoles (Beta vulgaris L.). Measurements with vacuoles in their suspension medium gave V_vac = 10 ± 2 millivolts (referred to the external medium) when 3 molar KCl microelectrodes were used. Buffering the microelectrode filling solution at pH 7.7 reversed the sign of the potential: V_vac = −7 ± 2 millivolts. The magnitude of the potential fluctuations was lowered by dilution (5-1000 times) with the suspension medium containing components released by the cells during the mechanical preparation. Fluctuations were decreased by 50 millimolar KNO₃ while they were enhanced by 5 millimolar ATP-Mg. No noticeable change in membrane resistance was detected. The presence of an ATPase bound to the tonoplast may explain the recorded noise spectra. These spectra imply a close connection between the rate of ATPase functioning and the magnitude of ionic fluxes across the tonoplast. It is suggested that noise analysis could be used to detect ATPase (or related enzyme) activity in vacuoles. Possible use of H⁺ diffusion through a buffered microelectrode, to modify intravacuolar pH, is also suggested.     

Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Intracellular recordings from phycomyces

Intracellular recordings from the giant sporangiophore of Phycomyces stage II were obtained. The mean transmembrane potential for 30 observations was −119.9 millivolts (negative inside), and it did not change either as a result of a light stimulus or during dark adaptation. Injected depolarizing and hyperpolarizing step currents and steady currents did not produce any avidence of spike activity. We conclude that light transduction and dark adaptation in Phycomyces are not based on alterations of the transmembrane potential.     

Plasma membrane potential of the alga dunaliella, and its relation to osmoregulation

A fluorescent dye sensitive to membrane potential was used to follow the plasma-membrane potential in the unicellular halo-tolerant alga Dunaliella salina. The signal observed during dissipation of the plasma membrane potential by the addition of excess K⁺ and valinomycin, or a protonophore, was taken as a measure of the preexisting potential. A resting potential of −85 to −100 millivolts (negative inside) was calculated. Following a hypertonic shock, the plasma membrane was rapidly hyperpolarized. This hyperpolarization was transient, and the algae resumed their resting potential about 30 minutes after the shock. The resting plasma membrane potential was decreased by vanadate and is concluded to be generated mostly by the plasma membrane ATPase of Dunaliella. The transient hyperpolarization following a hypertonic shock indicates, therefore, a transient activation of the ATPase. This is further corroborated by a rapid transient decrease in the intracellular ATP following a hypertonic shock and its inhibition by vanadate. It is suggested that activation of the plasma membrane ATPase may be the trigger for osmoregulation in Dunaliella.     

Potassium Channels in Chara corallina: CONTROL AND INTERACTION WITH THE ELECTROGENIC H PUMP

Plasmalemma electrical properties were used to investigate K⁺ transport and its control in internodal cells of Chara corallina Klein ex Willd., em R.D.W. Cell exposure to solutions containing 10 mm KCl caused the potential, normally −250 millivolts (average), to depolarize in two steps. The first step was a 21 millivolt depolarization that lasted from 1 to 40 minutes. The second step started with an action potential and left the membrane potential at −91 millivolts, with a 10-fold reduction in resistance. We suggest that the second step was caused by the opening of K⁺ -channels in the membrane. This lowered the resistance and provided a current pathway that partially short-circuited the electrogenic pump. Although largely short-circuited, the electrogenic pump was still operating as indicated by: (a) the depolarized potential of −91 millivolts was more negative than Ek (=−42 millivolts in 10 mm K⁺); (b) a large net K⁺ uptake occurred while the cell was depolarized; (c) both the electrogenic pump inhibitor, diethylstilbestrol, and the sulfhydryl-reagent N-ethylmaleimide (which increased the passive membrane permeability) further depolarized the potential in 10 mm KCl.A two-phase recovery back to normal cell potentials occurred upon lowering the K⁺ concentration from 10 to 0.2 mm. The first phase was an apparent Nernst potential response to the change in external K⁺ concentration. The second phase was a sudden hyperpolarization accompanied by a large increase in membrane resistance. We attribute the second phase to the closing of K⁺ -channels and the removal of the associated short-circuiting effect on the electrogenic pump, thereby allowing the membrane to hyperpolarize. Further experiments indicated that the K⁺ -channel required Ca²⁺ for normal closure, but other ions could substitute, including: Na⁺, tetraethylammonium, and 2,4,6-triaminopyrimidine. Apparently, K⁺ -channel conductance is determined by competition between Ca²⁺ and K⁺ for a control (gating?) binding site.     

Phytosiderophore release from nodal,primary, and complete root systems in maize

Some maize (Zea mays L.) hybrids grown in high pH soil in Nebraska suffer from severely reduced yields caused by iron (Fe) deficiency chlorosis. Hybrids which recover from early season Fe-deficiency chlorosis and yield well are termed Fe-efficient or tolerant. Most Fe-efficient gramineous species respond to Fe-deficiency stress by releasing phytosiderophores (mugineic acid and its derivatives) into the rhizosphere, thereby increasing Fe availability and uptake of the Fe³⁺-phytosiderophore complex via a high affinity uptake system. Field-grown Fe-efficient maize recovers from Fe-deficiency chlorosis at a stage when nodal roots have become the dominant root system. Quantifying phytosiderophore release from hydroponically grown plants has been proposed as a viable alternative to time-consuming and variable field trials and has been used successfully to delineate among Fe-efficient and Fe-inefficient lines of oat (Avena sativa L.) and wheat (Triticum aestivum L.). Our objectives were (1) to determine if phytosiderophore release differed between nodal- and primary-root systems of maize, and (2) to compare phytosiderophore release from 12 hybrids. Root exudates secreted during daily 4-h collections were analyzed for their Fe-solubilizing ability, which was equated to phytosiderophore release. Nodal root systems released significantly more phytosiderophore than primary- or complete-root systems. In early experiments, an Fe-efficient hybrid (P3279) released more phytosiderophore from nodal roots than an Fe-inefficient hybrid (P3489). Tests of an additional 10 hybrids showed that phytosiderophore release varied significantly among the cultivars but did not clearly distinguish between hybrids classified as Fe-efficient or Fe-inefficient in individual company trials. We recommend using nodal roots when studying Fe-stress response mechanisms in maize.     

Proton extrusion by wheat roots exhibiting severe aluminum toxicity symptoms

The mechanisms of Al rhizotoxicity are not known, but disruption of membrane function has been a persistent hypothesis. The objective of this study was to establish whether cells of Al-cultured wheat roots (Triticum aestivum L. cv Tyler) exhibiting severe Al toxicity symptoms were capable of vigorous proton extrusion. The membrane electrical potential difference (E_m) was measured in individual cells throughout the first centimeter of root tips during perfusion with Al solutions similar to or more concentrated than those of the culture medium. For both Al-cultured and control roots the resting E_m was −100 millivolts, and 1 millimolar acetic acid induced cyanide-sensitive hyperpolarizations to −180 millivolts at a maximum rate of −30 millivolts per minute. Al, like Ca²⁺, enhanced the negativity of the E_m of cells already treated with acetic acid. Both acetic acid and fusicoccin stimulated net proton extrusion from Al-cultured and control roots, both of which also extruded protons in the absence of these stimulants. These results demonstrate that wheat roots exhibiting severe Al toxicity symptoms had an undiminished capacity to extrude protons, that the membranes were intact, and that ATP synthesis was sufficient to supply the proton-translocating ATPases.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): IV. Correlation between Membrane Effects and Resistance to Graminicides

The herbicidally active aryloxyphenoxypropionates diclofop acid, haloxyfop acid, and fluazifop acid and the cyclohexanedione sethoxydim depolarized membranes in coleoptiles of eight biotypes of herbicide-susceptible and herbicide-resistant annual ryegrass (Lolium rigidum). Membrane polarity was reduced from −100 millivolts to −30 to −50 millivolts. Membranes repolarized after removal of the compounds only in biotypes with resistance to the compound added. Repolarization was not observed in herbicide-susceptible L. rigidum, nor was it observed in biotypes resistant to triazine, triazole, triazinone, phenylurea, or sulfonylurea herbicides but not resistant to aryloxyphenoxypropionates and cyclohexanediones. Chlorsulfuron, a sulfonylurea herbicide, at a saturating concentration of 1 micromolar, reduced membrane polarity in all biotypes studied by only 15 millivolts. The recovery of membrane potential following the removal of chlorsulfuron was restricted to chlorsulfuron-susceptible and -resistant biotypes that did not exhibit diclofop resistance. These differences in membrane responses are correlated with resistance to dicloflop rather than with resistance to chlorsulfuron. It is suggested that the differences may reflect altered membrane properties of diclofop-resistant biotypes. Further circumstantial evidence for dissimilarity of properties of membranes from diclofop-resistant and diclofop-susceptible ryegrass is provided by observations that K⁺/Na⁺ ratios were significantly higher in coleoptiles from diclofop-resistant biotypes than in coleoptiles from susceptible plants. Intact and excised roots from susceptible biotypes were capable of acidifying the external medium, whereas roots from resistant biotypes were unable to do so. The ineluctable conclusion is that in L. rigidum the phenomena of membrane repolarization and resistance to aryloxyphenoxypropionate and cyclohexanedione herbicides are correlated.     

A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince,pear and olive genotypes under iron deficiency stress

Quince (Cydonia oblonga Mill.), pear (Pyrus communis L.) and olive (Olea europaea L.) genotypes were evaluated for their tolerance to iron deficiency stress by growing young plants in three types of aerated nutrient solutions: (1) with iron, (2) without iron or (3) low in iron and with 10 mM bicarbonate. Plants were obtained either from rooted softwood cuttings or from germination of seeds. The degree of tolerance was evaluated with several indices: (1) the chlorophyll content, (2) the root Fe³⁺ reducing capacity and (3) the whole plant relative growth. Fifteen hours before Fe³⁺ reducing capacity determination, iron was applied to the roots of plants with iron-stress, since this method resulted in increasing the reductase activity. All quince and pear genotypes increased the root Fe³⁺ reducing capacity when grown in the treatments for iron-stress, in relation to control plants of the same genotypes. In olive cultivars, the Fe³⁺ reducing capacity was lower in the iron-stress treatments than in the control one. Studying the relationship between relative growth and chlorophyll content for each genotype under iron-stress, in relation to both indices in control plants, a classification of species and genotypes was established. According to that, most olive cultivars and some pear rootstocks and cultivars appear more iron-efficient than quince rootstocks. Our study shows that in some woody species, determining root Fe³⁺ reducing capacity is not the best method to establish tolerance to iron deficiency stress.     

Active extrusion of protons into deionized water by roots of intact maize plants

The investigations were focussed on the question as to whether roots of intact maize plants (Zea mays L. cv Blizzard) release protons into deionized H₂O. Plants in the six to seven leaf stage depressed the pH of deionized H₂O from 6 to about 4.8 during an experimental period of 4 hours. Only one-third of the protons released could be ascribed to the solvation of CO₂ in H₂O. The main counter anions released were Cl⁻, NO₃⁻, and SO₄²⁻. At low temperature (2°C), the H⁺ release was virtually blocked while a relatively high amount of K⁺ was released. The presence of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the external solution increased the H⁺ secretion significantly. Addition of vanadate to the outer medium inhibited the H⁺ release while fusicoccin had a stimulating effect. Substituting the nutrient solution of deionized H₂O resulted in a substantial increase of the membrane potential difference from −120 to −190 millivolts. The experimental results support the conclusion that the H⁺ release by roots of intact maize plants is an active process driven by a plasmalemmalocated ATPase. Since the net H⁺ release was not associated with a net uptake of K⁺, it is unlikely to originate from a K⁺/H⁺ antiport.     

Electrical evidence for turgor inhibition of proton extrusion in sugar beet taproot

Sections of sugar beet (Beta vulgaris L.) taproot were incubated in various concentrations of mannitol. At 0.4, 0.6, and 0.8 molar, the membrane electrical potential difference (E_m) averaged about −130 millivolts; at 0.2 molar, about −90 millivolts; and at 0 molar, between −60 and −80 millivolts. Additions of 10 millivolts acetate to the incubation solutions (all at pH 5) enhanced the membrane polarity to about −200 millivolts. We conclude from these and previous findings that high turgor inhibits proton extrusion in the sugar beet, but that proton extrusion can be activated in fully turgid tissue by acidification of the cytoplasm. A possible function of this turgor effect may be the control of turgor itself.     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Perturbation of Chara Plasmalemma Transport Function by 2[4(2',4'-Dichlorophenoxy)phenoxy]propionic Acid

Electrophysiological measurements on internodal cells of Chara corallina Klein ex Willd., em. R.D.W. revealed that in the presence of (2-[4-(2′,4′-dichlorophenoxy)phenoxy]propionic acid) (diclofop) the membrane potential was very sensitive to the pH of the bathing medium. At pH 5.7, 100 micromolar diclofop caused a slow reduction in the electrogenic component of the membrane potential to the value of −123 ± 5 millivolts. Membrane resistance initially decreased, recovered transiently, then stabilized at approximately 65% of the control value. At pH 7.0, the potential appeared to plateau around −200 millivolts before rapidly declining to −140 ± 4 millivolts; removal of diclofop resulted in recovery of the electrogenic component. Diclofop reduced cytoplasmic ATP levels by 96.4% and 36.6% at pH 5.7 and 7.0, respectively. At pH 8.2, diclofop did not change the ATP concentration significantly, but induced a hyperpolarization of the membrane potential to near −250 millivolts, and also reduced or inhibited the dark-induced hyperpolarization; the light-induced depolarization was reduced to a lesser extent. DCMU applied in the light elicited the same response at the plasmalemma as placing cells in the dark. When K⁺ channels were opened and cells depolarized with 10 millimolar K⁺, diclofop induced a further depolarization of approximately 30 millivolts. Cells decoupled with HPO₄⁻² were still sensitive to diclofop. Currents associated with OH⁻ efflux and HCO₃⁻ influx, as measured with a vibrating probe technique, became spatially destabilized and reduced in magnitude in the presence of diclofop. After 60 minutes, most of the cell surface was engaged in a low level of OH⁻ efflux activity. The results indicate that diclofop may be a proton ionophore at pH 7.0 and 5.7. At pH 8.2, diclofop may inhibit the operation of the H⁺-ATPase and OH⁻ efflux systems associated with HCO₃⁻ transport by perturbing the control processes that integrate the two, without a reduction in ATP concentration.     

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (± standard deviation) for unplasmolyzed tobacco cells was −52 ± 12 millivolts, for cells in 0.3 molar mannitol, −50 ± 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, −49 ± 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was −49 ± 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Dependency of Iron Reduction on Development of a Unique Root Morphology in Ficus benjamina L

The activity of the Fe³⁺ reductase of excised adventitious roots of Ficus benjamina L., grown in hydroponic culture without iron, was determined by a colorometric assay simplified by the use of a microplate reader. Reductase activity remained the same from pH 4.5 to 6.5 and decreased sharply above pH 6.5. Acetate buffer inhibited reduction. During early stages of root growth, excised roots did not exhibit Fe³⁺ reductase activity. After several weeks and extensive root system development, Fe³⁺ reduction still was not detectable in primary roots, but intermediate and high rates of reduction occurred in lateral and newly formed root clusters, respectively. Clustered roots only developed on plants grown at 0 or very low (<1 micromolar) iron. Microscopic examination revealed the root cluster to be composed of up to 30 lateral roots, usually less than 1 millimeter in diameter and 1 centimeter in length, that were completely covered with root hairs.