Propagated fluctuations of the electric potential in the apoplasm of Lepidium sativum L. roots

The electric potential on the surface of the Lepidium sativum L. root apex was recorded by means of six non-polarizable electrodes. Nonevoked fluctuations of the potential with amplitudes below 0.1 mV were observed. The fluctuations could be reversibly inhibited either by ether vapor or by anoxia caused by N₂. They did not occur in killed roots. Cross-correlation analysis of the fluctuations from six electrodes located one above another along the 3-mm apical region showed a pattern of time delay which indicates that the fluctuations may be the consequence of signals propagated in the root with a velocity of 3–9 mm · s^–1 in a basipetal direction from the root cap. We hypothesize that the fluctuations are due to signals of an unknown nature propagated along an intrasymplasmic continuous system, the symreticulum, composed of the cortical ER of individual cells and desmotubules passing through the plasmodesmata.Abbreviations AC alternating current - AP action potential - ACF autocorrelation function - CCF cross-correlation function - DC direct current - EEP extracellular electric potential This research was supported by Bundesminister für Forschung und Technologie, Bonn, and Ministerium für Wissenschaft und Forschung, Düsseldorf, (AGRAVIS). We are grateful to Mr. Dipl.-Ing. P. Blasczyk for constructing the amplifiers and for advice in instrumentation, and to Mr. H. Laubach for constructing the mechanical assembly.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

The coupling between extra- and intracellular electric potentials in Bidens pilosa L.

The extracellular electrical potential in Bidens pilosa has been compared with the transmembrane potential in hypocotyl cells. We found that variations in the extracellular electrical potential, induced by different KCI concentrations, were identical to the variations of the transmembrane potential. In order to explain the strong correlation between the extracellular potential and the transmembrane potential, an interpretative model based on the electrical diagram of the plant was tested. From our data, we found that: (1) the symplast behaves as a low resistance pathway compared with the apoplast; and (2) the cell wall contributes weakly (20% at the most) to the transmembrane potential. It is concluded that each hypocotyl cell is strongly coupled electrically with its neighbour. This property, together with the high apoplast resistance, accounts for the similarity in the extracellular and transmembrane potential measurements. These properties, which are characteristic of excitable organs, support the existence of action potentials in Bidens.     

Transmembrane electrical potentials in growing maize roots

The anti-auxin 4-chlorophenoxyisobutyric acid (PCIB) applied at a concentration of 10^-2 mol m^-3 to maize root segments was found to induce a transmembrane electrical potential of up to-130 mV (pd of 30 mV). The kinetics of this response were comparable to the time scale for PCIB-stimulated H⁺-extrusion. Both effects are eliminated by the addition of p-fluoromethoxycarbonyl cyanide phenylhydrazone (FCCP). Treatment with fusicoccin (FC) and PCIB together does not result in a hyperpolarization greater than with FC alone. Benzoic acid (10^-2 mol m^-3) had no effect on the transmembrane electrical potentials. These results are discussed in relation to a possible electrogenic proton pump which may be regulated by perturbations in the cellular auxin content or activity.Abbreviations ATPase adenosine triphosphatase - FC fusicoccin - FCCP p-fluoromethoxy carbonylcyanide phenylhydrazone - IAA indole-3yl-acetic acid - NAA naphthyl-lylacetic acid - PCIB 4-chlorophenoxyisobutyric acid - PD potential difference     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm · s^–1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a slow-wave potential. These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.Abbreviations and Symbols GR growth rate - P_x xylem pressure - R_in cell input resistance - SWP slow wave potential - V_m membrane potential - V_s surface potential This work was supported by grants to D.J.C. from the National Science Foundation and the U.S. Department of Energy.     

Plasticity of the electric organ discharge waveform of the electric fish Brachyhypopomus pinnicaudatus I. Quantification of day-night changes

The electric organ discharge of the gymnotiform fish Brachyhypopomus pinnicaudatus is a biphasic waveform. The female's electric organ discharge is nearly symmetric but males produce a longer second phase than first phase. In this study, infrared-sensitive video cameras monitored the position of unrestrained fish, facilitating precise measurement of electric organ discharge duration and amplitude every 2 h for 24 h. Males (n=27) increased electric organ discharge duration by 37 ± 12% and amplitude by 24 ± 9% at night and decreased it during the day. In contrast, females (n=8) exhibited only minor electric organ discharge variation over time. Most of a male's increase occurred rapidly within the first 2–3 h of darkness. Electric organ discharge values gradually diminished during the second half of the dark period and into the next morning. Modulation of the second phase of the biphasic electric organ discharge produced most of the duration change in males, but both phases changed amplitude by similar amounts. Turning the lights off at mid-day triggered an immediate increase in electric organ discharge, suggesting modification of existing ion channels in the electric organ, rather than altered genomic expression. Exaggeration of electric organ discharge sex differences implies a social function. Daily reduction of duration and amplitude may reduce predation risk or energy expenditure. Accepted: 12 September 1998     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997     

Effects of white,blue, red light and darkness on pH of the apoplast in the Samanea pulvinus

Leaflet movements in Samanea saman (Jacq.) Merrill are driven by fluxes of K⁺, anions, and water through membranes of motor cells in the pulvinus (R.L. Satter et al., 1974, J. Gen. Physiol. 64, 413–430). Extensor cells take up K⁺ and swell in white light (WL) while flexor cells take up K⁺ and swell in darkness (D). Excised strips of extensor and flexor motor tissue acidify their bathing medium under conditions that normally promote increase in K⁺ in the intact tissue, and alkalize the medium under conditions that normally induce decrease in K⁺ (A. Iglesias and R.L. Satter, 1983, Plant Physiol. 72, 564). To obtain information on pH changes in the whole pulvinus, we measured effects of light on pH of the apoplast, using liquid membrane microelectrodes sensitive to H⁺. We report the following: (1) The pH of the extensor apoplast was higher than that of the flexor apoplast in WL and in D (pH gradient of 1.0 units in WL and 2.0 units in D). Apoplastic pH might affect K⁺ transport through the plasma membranes of Samanea motor cells, since the conductance, gating, and selectivity of ionic channels in other systems depend upon external pH. (2) Extensor cells acidified and flexor cells alkalized their environment in response to irradiation with WL, while the reverse changes occurred in response to D. These results are consistent with the results of Iglesias and Satter (1983), and support the physiological relevance of data obtained with excised tissue. (3) The pH changes in response to irradiation with red light were similar to those obtained with D; also, the pH changes in response to blue light were similar to those obtained with WL. The pulvinus closed in red light as in darkness and opened in WL, but failed to open in blue light. The advantages and limitations of apoplastic pH measurements for assaying H⁺ transport are discussed.Abbreviations BL blue light - D darkness - RL red light - WL white light     

Active electric properties of cardiac muscle

A model of electrical activity in the heart has been developed that treats the intracellular domain and the extracellular domain as electrical syncytia with anisotropic resistivities (bi-syncytial model). At the microscopic level, propagation is assumed to proceed primarily along the axes of individual cells. Considerations at the macroscopic level relate the transmembrane current to the intracellular and extracellular resistivity and the transmembrane potential. The result is a relationship between instantaneous extracellular potentials and cardiac action potentials.     

Regulation of sucrose-phosphate-synthase activity in spinach leaves by protein level and covalent modification

A dot-blot technique was developed using monoclonal antibodies to measure, rapidly and accurately, the amount of sucrose-phosphate synthase (SPS; EC 2.4.1.14) protein present in a crude extract from spinach (Spinacia oleracea L. cv. Dark Green Bloomsdale) leaves; this was compared with SPS activity in this material. During leaf development, increased SPS activity followed closely the increase in enzyme-protein level, indicating denovo synthesis or altered turn-over rates for SPS. In contrast, activation of SPS by illumination of leaves or by mannose treatment of leaf discs in the dark (M. Stitt et al. Planta 174, 217–230) occurred without a significant change in the level of enzyme protein. Since conditions which altered SPS activity did not affect immunoprecipitation or mobility of the 120-kilodalton (kDa) subunit of the enzyme during denaturing gel electrophoresis, some form of protein modification other than proteolysis must be involved. Overall, the results indicate that regulation of SPS activity can involve changes in the level of enzyme protein and-or covalent modification.Abbreviations kDa kilodalton - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - SPS sucrosephosphate synthase Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Reseach Service, Raleigh. Paper No. 11789 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA     

Differences in the electric birefringence of spectrin dimers and tetramers as shown by the fast reversing electric pulse method

The electric birefringence of purified Spectrin has been examined in medium of low ionic strength ai 20°C and for electric fields smaller than 4 × 10⁴ V m^?1. using the reversing electric pulse method. This technique allows study of the permanent and induced dipole electric moment of macromolecules more easily than in measurements using only rectangular pulses. We show that spectrin heterodimers and heterotetramers have different electro-optical properties. The relaxation time of the tetramer (7 μs) is significantly longer than that of the dimer (4.5 μs). Tetramers and dimers have also different polarizability parameters.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

Photosynthetic and structural acclimation to light direction in vertical leaves of Silphium terebinthinaceum

The azimuth of vertical leaves of Silphium terebinthinaceum profoundly influenced total daily irradiance as well as the proportion of direct versus diffuse light incident on the adaxial and abaxial leaf surface. These differences caused structural and physiological adjustments in leaves that affected photosynthetic performance. Leaves with the adaxial surface facing East received equal daily integrated irradiance on each surface, and these leaves had similar photosynthetic rates when irradiated on either the adaxial or abaxial surface. The adaxial surface of East-facing leaves was also the only surface to receive more direct than diffuse irradiance and this was the only leaf side which had a clearly defined columnar palisade layer. A potential cost of constructing East-facing leaves with symmetrical photosynthetic capcity was a 25% higher specific leaf mass and increased leaf thickness in comparison to asymmetrical South-facing leaves. The adaxial surface of South-facing leaves received approximately three times more daily integrated irradiance than the abaxial surface. When measured at saturating CO₂ and irradiance, these leaves had 42% higher photosynthetic rates when irradiated on the adaxial surface than when irradiated on the abaxial surface. However, there was no difference in photosynthesis for these leaves when irradiated on either surface when measurements were made at ambient CO₂. Stomatal distribution (mean adaxial/abaxial stomatal density = 0.61) was unaffected by leaf orientation. Thus, the potential for high photosynthetic rates of adaxial palisade cells in South-facing leaves at ambient CO₂ concentrations may have been constrained by stomatal limitations to gas exchange. The distribution of soluble protein and chlorophyll within leaves suggests that palisade and spongy mesophyll cells acclimated to their local light environment. The protein/chlorophyll ratio was high in the palisade layers and decreased in the spongy mesophyll cells, presumably corresponding to the attentuation of light as it penetrates leaves. Unlike some species, the chlorophyll a/b ratio and the degree of thylakoid stacking was uniform throughout the thickness of the leaf. It appears that sun-shade acclimation among cell layers of Silphium terebinthinaceum leaves is accomplished without adjustment to the chlorophyll a/b ratio or to thylakoid membrane structure.     

Effects of blue and red light on unrolling of rice leaves

Unrolling of the second leaf of 8-day-old rice (Oryza sativa L.) seedlings was promoted by weak blue light (B), but not by red light (R). The effect of B was counteracted by irradiation with R just before or after the B. The counteracting effect of R was reversed by subsequent irradiation with far-red light but not by B, even if B was applied for 10 h. The B was effective when the region 0.5–2 cm from the tip of the leaf was irradiated. These results indicate that in rice photoreceptors for blue light located in the region 0.5–2 cm from the tip of the leaf play a key role in leaf unrolling and that a B-absorbing pigment and phytochrome participate in leaf unrolling in a closely related manner.Abbreviations B blue light - R red light - FR far-red light - W white light - D dark This work was presented at the Annual Meeting of the Japanese Society of Plant Physiologists on April 4, 1978, in Hiroshima     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996     

An ephaptic transmission model of CA3 pyramidal cells: an investigation into electric field effects

Extracellular electric fields existing throughout the living brain affect the neural coding and information processing via ephaptic transmission, independent of synapses. A two-compartment whole field effect model (WFEM) of pyramidal neurons embedded within a resistive array which simulates the extracellular medium i.e. ephapse is developed to study the effects of electric field on neuronal behaviors. We derive the two linearized filed effect models (LFEM-1 and LFEM-2) from WFEM at the stable resting state. Through matching these simplified models to the subthreshold membrane response in experiments of the resting pyramidal cells exposed to applied electric fields, we not only verify our proposed model’s validity but also found the key parameters which dominate subthreshold frequency response characteristic. Moreover, we find and give its underlying biophysical mechanism that the unsymmetrical properties of active ion channels results in the very different low-frequency response of somatic and dendritic compartments. Following, WFEM is used to investigate both direct-current (DC) and alternating-current field effect on the neural firing patterns by bifurcation analyses. We present that DC electric field could modulate neuronal excitability, with the positive field improving the excitability, the modest negative field suppressing the excitability, but interestingly, the larger negative field re-exciting the neuron back into spiking behavior. The neuron exposed to the sinusoidal electric field exhibits abundant firing patterns sensitive to the input frequency and intensity. In addition, the electrical properties of ephapse can modulate the efficacy of field effect. Our simulated results are qualitatively in line with the relevant experimental results and can explain some experimental phenomena. Furthermore, they are helpful to provide the predictions which can be tested in future experiments.     

Separation of cell enlargement and division in bean leaves

A method is presented for inducing cell enlargement in intact leaves and leaf strips of Phaseolus vulgaris L. without the complication of cell division. Primary bean leaves complete cell division and stop growing after 10 d in dim red light. Transfer to white light induces expansion (50% in 24 h) which is entirely the consequence of cell enlargement. Leaf strips from red-light-grown seedlings placed in white light and provided external solutes (10 mM KCl+10 mM sucrose) expand at the same rate as intact leaves in the light. This system makes possible future investigation of the mechanism of leaf cell enlargement.     

Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate