Some Studies on Root Membrane Potentials

Root membrane potentials were measured with two calomel electrodesby placing barley roots between two different potassium solutionsin which the electrodes dip. These potentials were found todepend on the type, concentration, and pH of the salt used.This is taken to indicate that the transport number of K inthe membrane is dependent on these factors. Furthermore, thepotential measurements as a function of pH and KCI concentrationallowed the determination of the point of zero charge of thebarley root which was found to be at pH 2–9.     

The relationship between sensitivity to 60-Hz electric fields and induced transmembrane potentials in plants root cells

Summary Growth rates and cell diameters were determined from 12 species of plant roots exposed to a 60-Hertz (Hz) electric field of 360 Volts per meter (V/m) in an aqueous inorganic nutrient medium [conductivity: 0.07–0.09 Siemens per meter (S/m)]. The degree of growth depression ranged from zero to nearly 100 percent of control. Cell diameters ranged from 13.5 to 31.8 µm as an averaged value for procambial, cortical, and meristem cells. Sensitivity to the electric field as determined by root growth rate reduction increased with increasing cell size. Sensitivity also increased with increase in 60 Hz induced transmembrane potentials; the transmembrane potential threshold for growth reduction was about 6.0 mV and the potential for near-complete cessation of growth was about 10–11 mV.Two different hypothetical mechanisms of action by which applied electric fields induce biological effects at the cellular level were tested. The two mechanisms pertain to different possible modes of action of applied electric fields: one mechanism postulates the involvement of the transmembrane field, the other mechanism postulates the tangential electric field as the important factor for inducing biological effects. The data support the transmembrane and not the tangential field mechanism. It is concluded that the effects observed are consistent with a membrane related mechanism and that there is a narrow range (a few mV) between threshold and debilitating induced membrane potentials.     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

Summary Seminal roots ofCucumis sativus andCucurbita maxima were exposed to 60 Hz electric fields of 100–500 Vm^–1 in a conducting aqueous inorganic growth medium. Root growth rates were measured to produce a dose-response relationship for each species. The species were selected for study because of their familial relationship, reported sensitivity to 60 Hz, 360 Vm^–1 electric fields, and differing average root cell sizes. The latter characteristic influences the magnitude of ELF membrane potentials induced by constant-strength applied electric fields, but does not affect the magnitude of the electric field strength tangent to the cell surface. The difference in average root cell size betweenC. sativus (smaller cells) andC. maxima (larger cells) was used to evaluate two alternate hypotheses that the observed effect on root growth is stimulated by [1] the electric field tangent to the cell surface, or (2) a field-induced perturbation in the normal transmembrane potential of the cells.The results of the dose-response relationship studies are qualitatively consistent with the hypothesis that the effect is elicited by induced transmembrane potentials. The smaller-celled roots showed a substantially higher response threshold [C. sativus; E ₀ ^TH 330 Vm^–1] than did the larger-celled species [C. maxima; E ₀ ^TH 200 Vm^–1]. At field strengths above the response thresholds in both species, the growth rate ofC. sativus roots was less affected than that ofC. maxima roots exposed to the same field strength.     

Effects of 60-Hz electric fields on cellular elongation and radial expansion growth in cucurbit roots

Serial longitudinal and transverse sections were prepared from roots of Cucumis sativus and Cucurbita maxima that had been exposed/sham-exposed to 60-Hz electric fields for 0-2 days. Field exposures were selected to produce a 10-20% or a 70-80% growth inhibition in whole roots of both species. Cortical cell length and diameter were measured using a microscope and eyepiece micrometer; measurements were conducted "blind." In both species, inhibition of cellular elongation was associated with exposure to electric fields (EF). Cellular radial expansion was apparently unaffected by exposure to electric fields. The diameters of radially unexpanded or fully expanded C. sativus cortical cells were about 25-30% smaller than those of comparable cells in C. maxima roots. Previous studies of the relationship between rates of root growth and applied EF strength showed that the response thresholds of C. sativus and C. maxima differed by a similar relative amount. These results are consistent with the postulate that EF-induced effects in roots are elicited by induced transmembrane potentials.     

Mechanisms of Aluminum Tolerance in Wheat : An Investigation of Genotypic Differences in Rhizosphere pH, K, and H Transport, and Root-Cell Membrane Potentials

Control of rhizosphere pH and exclusion of Al by the plasma membrane have been hypothesized as possible mechanisms for Al tolerance. To test primarily the rhizosphere pH hypothesis, wheat cultivars (Triticum aestivum L. `Atlas 66' and `Scout'), which differ in Al tolerance, were grown in either complete nutrient solution, or 0.6 millimolar CaSO₄, with and without Al at pH 4.50. A microelectrode system was used to simultaneously measure rhizosphere pH, K⁺, and H⁺ fluxes, and membrane potentials (E_m) along the root at various distances from the root apex. In complete nutrient solution, the rhizosphere pH associated with mature root cells (measured 10-40 millimeters from the root apex) of Al-tolerant `Atlas 66' was slightly higher than that of the bulk solution, whereas roots of Al-sensitive `Scout' caused a very small decrease in the rhizosphere pH. In CaSO₄ solution, no significant differences in rhizosphere pH were found between wheat cultivars, while differential Al tolerance was still observed, indicating that the rhizosphere pH associated with mature root tissue is not directly involved in the mechanism(s) of differential Al tolerance. In Al-tolerant `Atlas 66', growth in a CaSO<sub>4</sub> solution with 5 micromolar Al (pH 4.50) had little effect on net K<sup>+</sup> influx, H<sup>+</sup> efflux, and root-cell membrane potential measured in cells of mature root tissue (from 10-40 mm back from apex). However, in Al-sensitive `Scout', Al treatment caused a dramatic inhibition of K⁺ influx and both a moderate reduction of H⁺ efflux and depolarization of the membrane potential. These results demonstrate that increased Al tolerance in wheat is associated with the increased ability of the tolerant plant to maintain normal ion fluxes and membrane potentials across the plasmalemma of root cells in the presence of Al.     

Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane proton ATPase in soybean

We have examined growth, water status and gene expression in dark-grown soybean (Glycine max L. Merr.) seedlings in response to water deficit (low water potentials) during the first days following germination. The genes encoded the plasma membrane proton ATPase and two proteins of 28 kDa and 31 kDa putatively involved in vegetative storage. Water potentials of stems and roots decreased when 2-day-old seedlings were transferred to water-saturated air. Stem growth was inhibited immediately. Root growth continued at control rates for one day and then was totally inhibited when the normal root-stem water potential gradient was reversed.Expression of mRNA for the 28 kDa and 31 kDa proteins, measured independently using specific 3-end probes, occurred about equally in stems. However, only the mRNA for the 31 kDa protein was detected in roots and at a lower abundance than in stems. Low water potentials increased the mRNA only for the 28 kDa protein in stems and the 31 kDa protein in roots. This differential expression followed the inhibition of stem growth but preceded the inhibition of root growth.The expression of the message for the ATPase, measured using a probe synthesized from a partial oat ATPase clone, was low in stems and roots but there was a 6-fold increase at low water potentials in roots. The increase followed the inhibition of root growth. This appears to be the first instance of regulation of ATPase gene expression in plants and the first demonstration of differential expression of the 28 kDa, 31 kDa, and ATPase messages. The correlation with the differential growth responses of the stems and roots raises the possibility that the differential gene expression could be involved in the growth response to low water potentials.     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

The region of elongation in Cucumis sativus and Cucurbita maxima roots was marked at increasing distances from the apex to provide an analog of increasing cell size. These roots were exposed/sham-exposed to 60 Hz electric fields and the growth rates of the root segments measured. The growth rate effect magnitude varied with increasing distance from the root tip at constant field strength, and with increasing applied field strength. These results provide strong, qualitative support for the postulate that ELF transmembrane potential induction is involved in the stimulation of ELF electric field effects in the plant root model system.     

60 Hz electric field parameters associated with the perturbation of a eukaryotic cell system

Summary Roots ofPisum sativum were exposed for seven days to 60 Hz electric fields ranging from 70–430 V/m in an aqueous medium whose conductivity was approximately 0.07 mho/m. (Corresponding current densities in the exposure medium associated with these field strengths ranged from 0.5–3.0 mA/cm²). Control and exposed roots were grown concomitantly in the same tank whose growth medium was continuously circulated. Temperature in the exposure medium was held at a constant 19° C. All experiments were conducted double blind. Root growth rates were determined daily. No perturbations in root growth were observed with electric fields of 150 V/m; there was a slight effect at 360 V/m, and a pronounced decrease in growth rate occurred at 430 V/m. Root conductivities are comparable to that of the growth medium. Under conditions in which growth inhibition occurs, it is estimated that induced 60 Hz cell membrane potentials would be of the order of 3–8 mV.     

Electrophysiological responses of maize roots to low water potentials: relationship to growth and ABA accumulation

The maintenance of root elongation is an important adaptive response to low water potentials (psi(w)), but little is known about its regulation. An important component may be changes in root cell electrophysiology, which both signal and maintain growth maintenance processes. As a first test of this hypothesis, membrane potentials (E(m)) were measured within the cell elongation zone of maize (Zea mays L.) primary roots. Seedlings were grown in oxygenated solution culture, and low psi(w) was imposed by the gradual addition of polyethylene glycol. Cells hyperpolarized approximately 25 mV in response to low psi(w), and after 48 h resting potentials remained significantly hyperpolarized at psi(w) lower than -0.3 MPa compared with roots at high psi(w). Inhibitor experiments showed that the hyperpolarization was dependent on plasma membrane H(+)-ATPase activity. Previous work showed that accumulation of abscisic acid (ABA) is required for the maintenance of maize primary root elongation at low psi(w). To determine if the mechanism of action of ABA involves changes in root electrophysiology, E(m) measurements were made during long-term exposure to low psi(w). Steady-state resting E(m) were measured in regions in which maintenance of cell elongation was dependent on ABA accumulation (2-3 mm from the apex), or in which elongation was inhibited regardless of ABA status (6-8 mm from the apex). E(m) was substantially more negative in ABA-deficient roots specifically in the 2-3 mm region. The results suggest that set-points for ion homeostasis shifted in association with the maintenance of root cell elongation at low psi(w), and that ABA accumulation plays a role in regulating the ion transport processes involved in this response.     

Aluminium effects on transmembrane potential in cells of fibrous roots of sugar beet

The effect of aluminium (Al) on the electrical transmembrane potential of epidermal and outer cortical root cells of intact seedlings of sugar beet (Beta vulgaris L. cv. Monohill) was studied. The potential difference to the surrounding medium was recorded with microelectrodes inserted into the vacuoles (PD_v) and cytoplasm (PD_c) of intact roots. Both long-term effects of AlCl₃ (100, μM present during cultivation) and immediate effects of AlCl₃ (10, 50, or 100 μM present in the assay medium), were measured. The effect of Al was measured at pH 4.0, 5.0 and 6.5 in order to obtain information on the toxicity of different Al forms existing at different pH values. Low pH and/or the presence of AlCl₃ during cultivation caused large depolarizations of the PD_v. Since the immediate effect of 2,4-dinitrophenol (DNP) on the resting potential of cells from Al-cultivated plants was negligible, it is likely that Al affects the metabolic component of the transmembrane potential. Aluminium also had an immediate effect on the PD in root cells of plants cultivated without Al. Addition of 10 or 50 μM Al to the assay medium caused hyperpolarization of PD_v in the presence of 0.5 mM Ca²⁺ at all pH values studied, depolarization of PD_c at pH 6.5, and hyperpolarization of PD_c at lower pH. At 1 mM Ca²⁺, or in the presence of K⁺ (≥ 2 mM), however, the same Al concentrations had little effect on PD_c. The strongest depolarizing effects of 10 or 50 μM Al in short-term treatments were obtained at pH 6.5, and were probably due to the soluble species Al(OH)₃, which is more frequent at pH 6.5 than at a lower pH. Addition of 50 μM Al caused alkalinization of the root medium at pH 6.5, but not at pH 4.0. Therefore, it is possible that Al at pH 6.5 is bound to, or translocated across, the membrane without the accompanying hydroxide ions. It is likely that most of the Al is bound to the root cells, since removal of Al from the buffer surrounding the roots did not cause the changed PD values to return to the original values. Aluminium also interacts with effects of Ca²⁺ and K⁺ on the membrane potential, since changes in PD, induced by changes in concentrations of Ca²⁺ and K⁺ are different in the absence and presence of Al.     

Soil temperature and flooding effects on two species of citrus

Summary Rough lemon (Citrus jambhiri Lush.) and sour orange (C. aurantium L.) seedlings were grown at constant soil temperatures of 16, 24, and 33 C for 3 months. Shoot and root growth of rough lemon was greatest at 33 C while growth of sour orange was greatest at 24 C. There were no significant effects of soil temperature on shoot: root ratio, leaf water potential or stomatal conductance. The hydraulic conductivity of intact root systems of both species was highest when seedlings were grown at 16 C. Thus, acclimation through greater root conductivity at low soil temperature may have compensated for decreased root growth at 16 C and negated effects of soil temperature on plant water relations. Half the plants growing at each soil temperature were subsequently flooded. Within 1 week, the soil redox potential (Eh) dropped below zero mV, reaching a minimum Eh of –250mV after 3 weeks of flooded conditions. Flooded plants exhibited lower root conductivity, a cessation of shoot growth, lower leaf water potentials, lower stomatal conductances, and visual sloughing of fibrous roots. Decreases in root conductivity in response to flooding were large enough to account for the observed decreases in stomatal conductance.Florida Agricultural Experiment Stations Journal Series No. 4080.     

Electrotaxis of zoospores of Phytophthora palmivora at physiologically relevant field strengths

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. We show here that zoospores of the plant pathogen Phytophthora palmivora exhibit anodal electrotaxis in electrical fields ≥0.5 V m⁻¹ comparable in size to the physiological fields around roots. An experimental protocol for applying weak electrical fields and quantifying electrotaxis is described. In this system, zoospore suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of encysted zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. The electrotactic response saturated at fields above 10 V m⁻¹ was inhibited in media of osmotic strength below 400 Osmol m⁻³, was maximal at pH 7.5 and increased at high zoospore densities. These data suggest that electrotaxis may be a useful adjunct to chemotaxis in root targeting by zoospores.     

Inhibition and recovery of growth processes in roots of pisum sativum L. Exposed to 60-Hz electric fields

Roots of Pisum sativum L. were chronically exposed in aqueous inorganic nutrient medium to 60-Hz electric fields between 140 and 490 V/m (growth medium conductivity ～ 0.08 S/m). The growth rate, meristematic mitotic index, and growth rate recovery of the roots were determined. At 140 V/m there was no perturbation in growth rate or mitotic index. At 430 V/m the growth rate and the mitotic index were reduced. The mitotic index had a maximum depression (～ 55% of control), which occurred at 4 h. The depression in growth rate was immediate and constant over time. When roots were exposed to an electric field at 430 V/m for 2 days, the growth rate was depressed by about 40%. When the field was terminated, the growth rate steadily increased and was almost normal after 5 days. At 490 V/m root growth rate was almost completely arrested. According to these results, there is a narrow range of induced membrane potentials that span the range from slightly altered to almost completely arrested growth rates.     

Influence of soil water deficits on root growth of cotton seedlings

Summary Cotton (Gossypium hirsutum L. cv. H14) seedlings were raised in soil of differing soil water content in specially designed pots in which the roots had access to freely available water and nutrients located 2.5 cm below the base of the soil core. The time for root emergence from the soil core and the rate of root growth were measured daily from sowing to harvest. The root and shoot dry weight and leaf water potential were measured at the final harvest 16 days after sowing. As soil water content decreased, the root emerged from the soil earlier and the initial rate of root elongation was faster. In spite of the availability of freely available water, the plants in the soil at low water contents had significantly lower leaf water potentials than those in soil at high water contents. The root: shoot ratio increased as the soil water content decreased. This arose from an absolute increase in root weight, with shoot weight not being significantly affected.     

Plant growth, nutrient acquisition and mycorrhizal symbioses of a waterlogging tolerant legume (Lotus glaber Mill.) in a saline-sodic soil

Seedlings of Lotus glaberMill., were grown in a native saline-sodic soil in a greenhouse for 50 days and then subjected to waterlogging for an additional period of 40 days. The effect of soil waterlogging was evaluated by measuring plant growth allocation, mineral nutrition and soil chemical properties. Rhizobiumnodules and mycorrhizal colonisation in L. glaberroots were measured before and after waterlogging. Compared to control plants, waterlogged plants had decreased root/shoot ratio, lower number of stems per plant, lower specific root length and less allocation of P and N to roots. Waterlogged plants showed increased N and P concentrations in plant tissues, larger root crown diameter and longer internodes. Available N and P and organic P, pH and amorphous iron increased in waterlogged soil, but total N, EC and exchangeable sodium were not changed. Soil waterlogging decreased root length colonised by arbuscular mycorrhizal (AM) fungi, arbuscular colonisation and number of entry points per unit of root length colonised. Waterlogging also increased vesicle colonisation and Rhizobium nodules on roots. AM fungal spore density was lower at the end of the experiment in non-waterlogged soil but was not reduced under waterlogging. The results indicate that L. glaber can grow, become nodulated by Rhizobium and colonised by mycorrhizas under waterlogged condition. The responses of L. glaber may be related its ability to form aerenchyma.     

The gradient between pre-dawn rhizoplane and bulk soil matric potentials, and its relation to the pre-dawn root and leaf water potentials of four species

Uptake of soil water by plants may result in significant gradients between bulk soil and soil in the vicinity of roots. Few experimental studies of water potential gradients in close proximity to roots, and no studies on the relationship of water potential gradients to the root and leaf water potentials, have been conducted. The occurrence and importance of pre-dawn gradients in the soil and their relation to the pre-dawn root and leaf water potentials were investigated with seedlings of four species. Pre-germinated seeds were grown without watering for 7 and lid in a silt loam soil with initial soil matric potentials of -0.02, -0.1 and -0.22 MPa. Significant gradients, independent of the species, were observed only at pre-dawn soil matric potentials lower than -0.25 MPa; the initial soil matric potentials were -0.1 MPa. At an initial bulk soil matric potential of -0.22 MPa, a steep gradient between bulk and rhizoplane soil was observed after 7 d for maize (Zea mays L. cv. Issa) and sunflower (Helianthus annuus L. cv. Nanus), in contrast to barley (Hordeum vulgare L. cv. Athos) and wheat (Triticum aestivum L. cv. Kolibri). Pre-dawn root water potentials were usually about the same as the bulk soil matric potential and were higher than the rhizoplane soil matric potential. Pre-dawn root and leaf water potentials tended to be much higher than rhizoplane soil matric potentials when the latter were lower than -0.5 MPa. It is concluded that plants tend to become equilibrated overnight with the wetter bulk soil or with wetter zones in the bulk soil. Plants can thus circumvent negative effects of localized steep pre-dawn soil matric potential gradients. This may be of considerable importance for water uptake and growth in drying soil.     

Nitrate uptake by bean (Phaseolus vulgaris L.) roots under phosphate deficiency

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO₃ distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO₃ concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.     

Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields

Growth, mitotic index, and growth rate recovery were determined for Vicia faba L. roots exposed to 60-Hz electric fields of 200, 290, and 360 V/m in an aqueous inorganic nutrient medium (conductivity 0.07-0.09 S/m). Root growth rate decreased in proportion to the increasing strength; the electric field threshold for a growth rate effect was about 230 V/m. The induced transmembrane potential at the threshold exposure was about 4-7 mV. The mitotic index was not affected by an electric field exposure sufficient to reduce root growth rate to about 35% of control. Root growth rate recovery from 31-96% of control occurred in 4 days after cessation of the 360 V/m exposure. The results support the postulate that the site of action of the applied electric fields is the cell membrane.     

The plasma membrane H+‐ATPase AHA2 contributes to the root architecture in response to different nitrogen supply

In this study the role of the plasma membrane (PM) H⁺‐ATPase for growth and development of roots as response to nitrogen starvation is studied. It is known that root development differs dependent on the availability of different mineral nutrients. It includes processes such as initiation of lateral root primordia, root elongation and increase of the root biomass. However, the signal transduction mechanisms, which enable roots to sense changes in different mineral environments and match their growth and development patterns to actual conditions in the soil, are still unknown. Most recent comments have focused on one of the essential macroelements, namely nitrogen, and its role in the modification of the root architecture of Arabidopsis thaliana. As yet, not all elements of the signal transduction pathway leading to the perception of the nitrate stimulus, and hence to anatomical changes of the root, which allow for adaptation to variable ion concentrations in the soil, are known. Our data demonstrate that primary and lateral root length were shorter and lower in aha2 mutant lines compared with wild‐type plants in response to a variable nitrogen source. This suggests that the PM proton pump AHA2 (Arabidopsis plasma membrane H⁺‐ATPase isoform 2) is important for root growth and development during different nitrogen regimes. This is possible by controlling the pH homeostasis in the root during growth and development as shown by pH biosensors.     

Aluminium tolerance in triticale,wheat and rye as measured by root growth characteristics and aluminium concentration

Summary Screening large populations of plant species for Al tolerance requires simple and rapid tests. In this study, root characteristics of 12 cultivars of triticale (X Triticosecale, Witt Mack), wheat (Triticum aestivum L.), and rye (Secale cereale L.) were measured in nutrient solution with 0 or 6 ppm Al added. Aluminum injury to roots of triticale and wheat was characterized by decreases in root length, increases in the number of roots, and in Al-sensitive Redcoat and Arthur wheats by decrease in root weight. Root length and number of roots were correlated in triticale (r=−0.73^*) and in wheat (r=−0.85^*). Root length was also correlated with root weight in wheat (r=0.65^*); there was no relationship between the number of roots and weight. Differences in Al tolerance of cultivars of the three species were greater when the solution was adjusted to pH 4.8 only on the first day of the experiment than when pH was maintained at pH 4.8 throughout the growing period. Triticale and rye cultivars low in ability to increase solution pH gradually overcame Al toxicity by increasing the nutrient solution pH between 12 and 22 days. Aluminum sensitive triticale and wheat accumulated more Al in roots than tolerant cultivars when the solution pH was not adjusted daily; but no differences in Al accumulation were obtained between wheat cultivars at constant pH value. This study indicated that root length and number of roots can be reliably used for screening triticales for Al tolerance within 12 days of exposure to Al. Root length, Al concentration, and dry weight after 22 days of Al treatment were also reliable criteria for evaluating differential Al tolerances among triticale cultivars.