Membrane Potentials and Resistances of Excitable Cells in the Petiole and Main Pulvinus of Mimosa pudica

Resting potentials of excitable cells in the petiole and mainpulvinus of Mimosa pudica depended on the external concentrationsof potassium ([K^$]) and sodium ([Na^$]), and peaks of the actionpotentials depended on the external concentration of chloride([Cl^–]) in the excitable protoxylem cells of the petioleand the upper-half cells of the main pulvinus, which suggeststhat the action potential in these cells is a Cl^– spike.In the lower-half cells of the main pulvinus, the peak of theaction potential did not show a clear dependence on [K^$], [Na^$]or [Cl^–]. This implies that there is a decrease in theion selectivity of the membrane toward the peak of the actionpotential. Effective membrane resistances of the excitable protoxylemand phloem cells of the petiole, and the lower-half cells ofthe main pulvinus were 3.9?3.1 M (mean?SD, n=7), 9.4?8.2 M (n=5)and 5.0?2.9 M (n=15), respectively. The membrane resistanceof the lower-half cells of the main pulvinus decreased on itssudden bending, but not always. ¹ Present address: 1st Department of Physiology, Hamamatsu UniversitySchool of Medicine, Handa-cho 3600, Hamamatsu 431-31, Japan. (Received November 17, 1981; Accepted January 29, 1982)     

Oscillations of the Membrane Potential of Pulvinar Motor Cells In Situ in Relation to Leaflet Movements of Desmodium motorium

The ultradian rhythmic movement of the lateral leaflets of Desmodiummotorium is accompanied by rhythmic changes of the extra- andintracellular electrical potentials in the pulvinus, which aremeasured in situ in the pulvinus against the bathing solutionof the petiole. Extra- and intracellular potentials oscillatewith 180'b0 phase difference to each other, as shown by simultaneousmeasurements of both types of potentials in the abaxial partof the pulvinus. Light-induced changes of these potentials movein opposite directions. The in situ membrane potential of themotor cells of the pulvinus was calculated from the differencebetween the extra- and intracellular potentials. It was foundto oscillate between –136 and –36 mV, in phase withthe intracellular and inverse to the extracellular potential.The phase relationship between the leaflet movement rhythm andthe in situ membrane potential rhythm was as follows: downwardmovement is preceded and accompanied by a strong depolarization,upward movement by hyperpolarization. Our results suggest that membrane depolarization in pulvinarmotor cells of Desmodium motorium drives and controls potassiumefflux and hyperpolarization potassium influx via potassiumchannels. Key words: Desmodium pulvinus, leaf movement, pulvinar motor cells, electrical potential     

Comparative Histocytology of the Petiole and the Main Pulvinus in Mimosa pudica L.

In Mimosa pudica, the main pulvinus, which brings about leafmovements, presents unusual structural characteristics in comparisonwith the petiole. Peculiar cellular features which exist inthe cortex, epidermis, parenchyma and endodermal regions includethe shape of the cells, their disposition and the location ofthe organelles. The central cylinder of the petiole is surrounded only by afew parenchyma layers whereas the central cylinder of the pulvinusforms a narrow central core enclosed in numerous cortical parenchymalayers. The phloem of the pulvinus contains collenchymatouscells towards the outside and possesses companion cells withwall ingrowths; these phloem members do not exist in the petiole.Xylem and protoxylem parenchyma cells of the petiole possesswall ingrowths which do not occur in homologous cells of thepulvinus. Moreover the pith of the pulvinus is composed of smallfibriform elements similar to the xylem fibriform elements ofthe organ. The structures observed may facilitate exchanges between cellsin the petiole and in the pulvinus. The predominant functionsof the organs relative to lateral and longitudinal transferof nutrients and conduction of stimuli are discussed. Mimosa pudica L., sensitive plant, pulvinus, ultrastructure, conduction of stimuli, leaf movement     

Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery

A decrease in electric resistance and an increase in the extracellularCl^– concentration ([Cl^–]) in the main pulvinus ofMimosa occurred immediately after the action potential of themotor cells. The beginnings of both changes were almost coincidentalwith the beginning of the rapid movement. A remarkable increasein [Cl^–] was seen in the lower half of the pulvinus, butonly a slight increase in the upper half. Release of Cl^–,probably with K⁺; and other ions, from the motor cells impliesan ejection of liquid from the vacuole. When recovery of thepulvinus following rapid movement was fast in light, [Cl^–]decreased to its initial level within 20 min. When the recoverywas slow in darkness, [Cl^–] decreased at a slow rate andmaintained a higher level than its initial one for a long time.Photosynthetic inhibitors delayed recovery and the decreasein [Cl^–] even in light. These facts suggest that the re-entryof ions into motor cells during recovery partially requiresa photosynthetic energy supply. (Received February 12, 1980; )     

Analysis and Significance of Gravity-induced Asymmetric Growth in the Grass Leaf-sheath Pulvinus

The negative gravitropic response in the grass leaf-sheath pulvinusis a consequence of cell elongation involving all cells exceptthose of the uppermost region of the upper flank of an horizontallyoriented pulvinus. The lowermost layer of cells elongate maximally,and the regions in between elongate to intermediate extents.The resulting curvatures of a responding pulvinus can be expressedmathematically by relating the angle of curvature () to theoriginal length (L₀) and the maximal length of the lower surface(L₁) and the diameter of the organ (D), using the equation, = (L₁–L₀)/D, where is in radians. The elongation response(S) of any individual cell within the pulvinus can be expressedby the equation, S = 0.5-r cos, where r is the radius of thepulvinus and is in degrees. Microscopic measurement of celllengths in different regions of the pulvinus supports the mathematicalpredictions. Indirect support is also obtained from the useof colchicine, coumarin, dichloro-benzonitrile (DCBN) and isopropylN-chlorophenyl carbamate which exaggerate the inherent asymmetryduring gravitropic response. Coumarin and DCBN also induce thickeningsin the radial walls which appear first in the statenchyma, andlater, in cells located towards the outer periphery of the pulvinus.The distribution patterns of these thickenings suggest thatthe asymmetric growth response of the pulvinus may be due toa differential and radial, centrifugal transport of growth promotorsfrom the central statenchyma region. Gravity perception, grass pulvinus     

Effects of Inhibitors on the Light-Induced Changes in Electric Potential in Pulvinar Motor Cells of Phaseolus vulgaris L.

To analyze the mechanism of the light-induced changes in electricpotential in motor cells of the pulvinus of Phaseolus vulgarisL., inhibitors were applied to the pulvinus by the xylem perfusionmethod. The membrane potential was –60 to –80 mV,which indicated that the polarization was less than that ofcells of a pulvinus in air. A pulse (30 s) of blue light (BL)induced transient depolarization of the membrane in the motorcells. Red light (RL) caused hyperpolarization of the membrane.The magnitude of BL pulse-induced transient depolarization wasgreater under the hyperpolarized state caused by the RL. The membrane was depolarized to –30 to –40 mV onperfusion with the respiratory inhibitor NaN₃ (1 mM) and a pulseof BL or irradiation with RL did not cause any change in thepotential in the depolarized state. Hyperpolarization of themembrane by RL was inhibited by perfusion with DCMU (50 µM),an inhibitor of electron transport in photosynthesis. However,the magnitude of the depolarization induced by the pulse ofBL was not affected. Perfusion with a proton ionophore CCCP(100µM) depolarized the membrane and no change in thepotential was induced by a pulse of BL or by RL in the depolarizedstate. The extent of the BL pulse-induced depolarization of the membranewas proportional to the magnitude of the membrane potentialat the time of which the pulse of BL was applied. It is suggestedthat the active component of the membrane potential was inhibitedby the pulse of BL. The experimental results further supportthe hypothesis that BL inhibits the activity of the proton ATPaseand, thus, causes loss of the electrogenic component of themembrane potential of the pulvinar motor cells. (Received June 22, 1992; Accepted August 24, 1992)     

Action potential and rapid movement in the main pulvinus ofMimosa pudica

Relationships between action potential and rapid bending movement in the main pulvinus ofMimosa pudica L. were studied. The pulvinar action potential consisted of a fast-rising spike of 40 to 70 mV and a long-lasting plateau. It had a nature of propagating in both basipetal and acropetal directions at the rate of about 4 cm/sec. The rapid bending movement occurred 10 to 20 msec after the pulvinar action potential. The action potential and movement were both found only in the lower half of the main pulvinus, no response being observed in the upper half. The results suggested that the excitable cells which elicit the action potential as well as the rapid contraction exist only in the lower half of the pulvinus. Some possible mechanisms concerning the coupling between the electrical excitation and the contraction are discussed. This work was supported by the Grant in Aid for Scientific Research of the Ministry of Education.     

Diurnal Rb+ Transport from Roots to the Laminar Pulvinus in Phaseolus vulgaris L.

The primary leaves of kidney bean (Phaseolus vulgaris L.) openunder light and close in the dark by the deformation of thepulvinus resulting from diurnal distribution changes of K⁺,Cl^–, organic acid (or H⁺) and NO₃^–. When Rb⁺ was added as a tracer of K⁺ to the seedlings throughtheir roots, it was transported to the pulvinus cells duringthe light period but not during the dark period. Transpirationoccurred vigorously in the light but almost stopped in the dark.We concluded that Rb⁺ absorbed by the roots was carried to thepulvinus by the transpiration stream. Phaseolus vulgaris L., pulvinus, Rb⁺, diurnal transport transpiration stream     

Leaf Abscission in Soybean: Cytochemical and Ultrastructural Changes following Benzylaminopurine Treatment

6-benzylaminopurine (BAP) delays leaf abscission of soybeanGlycine max (L.) Merr. Abscission of the distal pulvinus ofprimary leaves was induced in 12-d-old seedlings or explantsby removal of the leaf blade. BAP applied to the cut end ofthe pulvinus following leaf blade removal delayed abscission.Discoloration of the pulvinus occurred before abscission commencedand the number of grana in chloroplasts within cortical parenchymacells of the pulvinus decreased over time following leaf bladeremoval. BAP prevented discoloration of pulvinus tissues anda decrease in grana number. Starch grains within amyloplastsof cells of the starch sheath in the pulvinus disappeared followingleaf blade removal, whereas starch accumulated within the abscissionzone prior to abscission. BAP prevented this apparent redistributionof starch and instead promoted an increase in starch withinplastids of cortical parenchyma cells of the pulvinus. Duringthe abscission process, cells within the separation layer enlargedand their nuclei and nucleoli became more evident prior to theirseparation from one another. Cell separation resulted from breakdownof middle lamellae and partial degradation of primary cell walls.Cycloheximide applied directly to the external surface of theabscission zone inhibited abscission in a similar way to theBAP treatment. These results suggest that BAP prevents abscissionby altering patterns of starch distribution in the pulvinusand abscission zone and by inhibiting the synthesis of proteinsthat typically appear de novo in induced abscission zone tissues. Key words: Benzylaminopurine, BAP, Soybean, Pulvinus, Abscission, amyloplast.     

Changes of the Apoplastic pH and K+ Concentration in the Phaseolus Pulvinus in situ in Relation to Rhythmic Leaf Movements

The apoplastic pH and K⁺ concentration of the extensor of thePhaseolus primary-leaf pulvinus in relation to rhythmic leafmovements have been investigated with double-barrelled ion-sensitivemicro-electrodes. Simultaneous measurements of leaf movementand ion activities in a fine hole of the extensor in situ showedco-existence of ultradian and circadian leaf movements as wellas of ultradian and circadian pH changes in the Water Free Space(WFS) of the extensor apoplast in situ. During circadian leafmovement the H⁺ and K⁺ activities in the WFS of the extensorchange in an antagonistic manner. When extensor cells swell(upward movement of the lamina) the H⁺ activity increases fromapproximately pH 6.7 to 5.9 and the K⁺ concentration decreasesfrom approximately 50 to 10 mol m^–3 and vice versa whenextensor cells shrink. These changes in the ionic activitiesin the WFS must be correlated with large changes in the ioncontent of the DFS and thus support the hypothesis that thecell walls of pulvinar cells serve as reservoirs for K⁺ andH⁺. Key words: Phaseolus pulvinus, apoplastic ionic activities, rhythmic leaf movements, ion-sensitive micro-electrodes (double-barrelled)     

Change in potassium distribution in a Phaseolus pulvinus during circadian movement of the leaf

Phaseolus moves its leaves upward and downward with circadianperiod. This movement of the leaf results from the differentialchange in the turgor on opposite sides of the pulvinus. Concentrations of K⁺, Na⁺, Mg⁺⁺, and Ca⁺⁺ in the upper and lowerhalves of the pulvinus and the water content of cells on bothsides of it were analyzed in relation to the deformation ofthe pulvinus. The results showed that (1) the pulvinus was deformedby expansion and contraction of the cells on its opposite sides;(2) among the four cations, the K⁺ concentration was markedlyhigh in both halves of the pulvinus; (3) the osmotic pressureof the upper and lower halves were nearly equal during the rhythmicdeformation of the pulvinus; (4) the expansion and contractionof the cells on the opposite sides of the pulvinus have a positivecorrelation only with a change in the K+ concentration expressedin terms of µmoles per mg protein; (5) the concentrationsof other cations such as Na⁺, Mg⁺⁺, Ca⁺⁺, expressed in termsof µmoles per mg protein, did not change during the circadiandeformation of the pulvinus. Thus, the rhythmic K⁺ movementseems to be the basis for pulvinar turgor movements. With respectto the mechanism of K⁺ movement, three possibilities are discussed. (Received November 7, 1975; )     

H+ tolerance of Chara Internodal Cells and Apparent Net Influx of H+ in Weakly Acidic Solutions: Implication of the Net Flux of H+ as a Minor Component among the Total Net Flux of Ions across the Intact Cell Membrane of Chara

Precise measurements of the net flux of protons in Chara internodalcells were made with a recently designed high-resolution pH-meter.Survival of intact Chara internodal cells in artificial pondwater (APW) that contained HC1 at various concentrations wasalso examined. The apparent net flux of H⁺ was inward and muchsmaller than that reported so far. In APW at pH 4.005, a valuehigher than the extracellular pH expected from the values ofH⁺ efflux reported to date, all of the intact Chara internodalcells died within a day. With reference to the data on the circadianflow of ions in the pulvinus of Phaseolus [Kiyosawa (1979) PlantCell Physiol. 20: 1621–1634, Hosokawa and Kiyosawa (1983)Plant Cell Physiol. 24: 1065–1072] and ionic regulationin Chara L-cells [Kiyosawa and Okihara (1988) Plant Cell Physiol.29: 9–19], a discussion is presented of the prossiblyminor contribution of the net flux of H⁺ in the generation ofthe electrical membrane potential. Regulation of the net fluxof H⁺ in weakly acidic APW is also discussed. (Received September 4, 1989; Accepted January 25, 1990)     

Excitable Channels in Nitellopsis obtusa as Part of an Active Ion-Transport System

The present paper is a study on the rapid and the slow excitablechannels of Nitellopsis obtusa. The working hypothesis is thatduring the excitation of these cells a Ca²⁺-dependent activeion-transport system in the plasmalemma is activated (Gyenesand Bulychev, 1979; Gyenes, Bulychev, and Kurella, 1980) whichmay interact with a light-dependent active transport systemalso present in the plasmalemma. It is found that under conditionsof maximal light-induced current changes, registered in voltageclamp experiments, the amplitudes of both action current componentsare relatively small (10–15 µA cm²) and they increaseup to 100–150 µA cm^–2 during 15–30 minin the dark. Cells may also be excited chemically under conditionsof unchanged voltage across the plasmalemma. It is suggestedthat in the excitation process of Nitellopsis obtusa two typesof ion channels take part/emdash electrically excitable passiveand chemically excitable active channels-both incorporated inone proteolipid complex of a Ca²⁺-dependent ATPase of the plasmalemma.     

Unequal distribution of potassium and anions within the Phaseolus pulvinus during circadian leaf movement

Ion and saccharide concentrations in the upper and lower partsof the laminar pulvinus of the primary leaf of Phaseolus vulgariswere measured in relation to the circadian movement. Concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, organic acid,NO₃^–, H₂PO₄^–, fructose and fructose-yielding saccharidesin the pulvinus were 75–120, 0.3–0.7, 5–8,6–12, 40–60, 60–73, 19–35, 2–9and 1–5 mM, respectively, and the osmotic pressure ofthe pulvinus was considered to be due to these ions. The cell volume in the expanding part was larger than that inthe contracting part. The change of the cell volume alteredthe molar concentration in the cell sap and therefore the amountof solutes actually transported from the upper to the lowerpart and vice versa was estimated from the concentration expressedin moles per gram of dry weight. Results showed that K⁺, Cl^–, organic acid (or H⁺) andNO₃^– moved from the upper to lower parts or vice versain the pulvinus in relation to its deformation, keeping theelectroneutrality among those ions, whereas C_a2⁺ and Mg²⁺ didnot move. The difference in the K⁺ concentration between theupper and lower parts when the leaf was up or down amountedto 30% of the whole osmotic pressure. This lead to the conclusionthat the endogenous clock-controlled unequal distribution ofK⁺, Cl^–, organic acid (or H⁺) and NO₃^– in the pulvinuscould be the force for the circadian leaf movement. (Received August 7, 1979; )     

Identification of the Excitable Cells in the Petiole of Mimosa pudica by Intracellular Injection of Procion Yellow

Excitable cells in the petiole of Mimosa pudica were locatedby microelectrode technique and stained with Procion YellowMx4R which was previously filled in the electrode and injectediontophoretically into the cells. Microscopic observations ofsections of the stained petioles revealed that protoxylem parenchymacells and narrow phloem cells were excitable. The protoxylemlocalized just inside the metaxylem was composed almost entirelyof the parenchyma cells which were 106.3±5.2 µmlong (mean±EM, n=15) and 14.2±0.6 µm indiameter (n =33). The excitable phloem cells were 76.4±4.1µm long (n=7) and 7.0±0.3 pan in diameter (n=37)and were thought to be companion cells or narrow parenchymacells or both. Amplitudes of action potentials recorded fromthe petiolar surface had a linear relation to those from theexcitable cells in the same petiole. From this fact and thearrangement of excitable cells in the petiole, we conclude thatwhen the transmission of action potential takes place in thepetiole all excitable cells in it are activated. ¹ Present address: 1st Department of Physiology, Hamamatsu UniversitySchool of Medicine, Handa-cho 3600, Hamamatsu 431-31, Japan. (Received September 7, 1982; Accepted November 8, 1982)     

Action Spectrum of Light Pulse-Induced Membrane Depolarization in Pulvinar Motor Cells of Phaseolus

In addition to circadian changes in the membrane potential andleaf movement, light applied to the pulvinus causes changesin both the membrane potential and the pulvinar movement inPhaseolus vulgaris L. Even after a short pulse of light, a transientdepolarization of the membrane occurs and leaf movement is observed.Decreases of turgor pressure of the motor cells are always precededby the depolarization. The direction of the leaf movement canbe explained by the decrease of turgor pressure in the motorcells on the irradiated side of the pulvinus. Using the OkazakiLarge Spectrograph at the National Institute for Basic Biology,we determined the action spectrum of the membrane depolarizationinduced by light pulses (30 s) in motor cells of Phaseolus.The pulvinus was left exposed to air during measurement of themembrane potential with microelectrodes. The action spectrumobtained was in the range of 300 to 730 nm. It had the highestpeak at 460 nm with lower peaks at 380 nm and 420 nm. Almostno sensitivity was observed at wavelengths shorter than 360nm and longer than 520 nm. Red and far-red light had no effecton the depolarization of the motor cell. The features of theaction spectrum are almost the same as those of the Blue-Typeresponse in plants. (Received January 9, 1997; Accepted February 14, 1997)     

Diurnal Changes in the Malic Acid Concentration in Phaseolus coccineus L. Pulvini

Concentration of malic acid was determined in pulvini and petiolesand in isolated parts of the pulvinus, i.e. extensor and flexorregions, in Phaseolus coccineus. In the light period of thecircadian cycle, the concentration of malic acid in whole pulvinireached the highest value of 35.1 mmole CW while in the darkphase the respective value was 21.0 mmole CW. In the petiole,the highest concentration of malic acid was only 15.3% of themaximum concentration in the whole pulvinus. In isolated regions of motor cells, a cyclic alternation inthe concentration of malic acid was observed. In the light phase,the maximum acid concentration of 43.7 mmole CW in the extensorzone corresponds with the lowest concentration of 15.5 mmoleCW in the flexor region. The lowest value of acid concentrationof 30.8 mmole CW in the extensor part corresponds with the highestacid concentration of 31.1 mmole CW in the flexor part in thedark phase. About 22% of the total concentration of malic acid was transportedbetween the two opposite parts of the pulvinus as dependingupon the phases of leaf movement. (Received February 28, 1986; Accepted May 23, 1986)     

Diurnal Geotropic responses of the Primary Leaves of Phaseolus vulgaris

When seedlings of Phaseolus vulgaris with leaves in the daytimeposition (almost horizontal to the ground) were turned upside-downduring the light period, their leaves moved upward away fromthe ground after about 20 min and ceased moving after about1.5 h. But when seedlings with leaves in the night time position(directed downward) were turned upside-down, their leaves moveddownward toward the ground after about 30 min and stopped movingabout 2 h later. Thus, Phaseolus primary leaves showed positiveor negative geotropic responses that correspohded to the darkor light period. This geotropic response of primary leaves was accompanied bythe redistribution of K⁺, Cl^– and NO₃- in the laminarpulvinus. These facts suggest that the circadian endogenousclock that is assumed to exist in Phaseolus vulgaris has atleast two regulation echanisms; one which measures time andanother which determines leaf postition in relation to gravityby changing the ion distribution in the pulvinus (Received February 12, 1983; Accepted May 17, 1983)     

Diurnal K+ and Anion Transport in Phaseolus Pulvinus

Diurnal movement of Phaseolus leaf is caused by deformationof the laminar pulvinus located at the joint of the leaf bladeand the petiole. The plants were cultured in solutions withvarious ion compositions, and changes of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃– and P₁ concentrations both in the upperand lower parts of the laminar pulvinus were measured. Culturein 10 mM KCl solution caused an increase in K⁺ and Cl^–concentrations both in the upper and lower parts without anysignificant change in the concentration of NO₃^–; culturein 10 mM KNO₃ solution caused an increase in K⁺ and NO₃^–concentration without any significant change in the concentrationof Cl^–; and culture in 10 mM KH₂PO₄ solution caused anincrease in K⁺ and P₁ concentrations without any significantchange in the concentrations of NO₃- and Cl^–. K⁺ moved from the upper to lower parts or from the lower toupper parts diurnally in all plants cultured in any solutionmentioned above. The main inorganic anion that accompanied thisK⁺ movement was Cl^– in KCl solution, and NO₃^– inKNO₃ solution. When the seedlings were cultured in distilledwater or in KH₂PO₄ solution, neither Cl^– NO₃^– norP₁ accompanied this K⁺ movement. In these cases, mainly H⁺ and/ororganic anions are supposed to move in exchange for and/or incombination with K⁺ movement. (Received November 8, 1982; Accepted June 13, 1983)     

Action Potential in the Trap-lobes of Aldrovanda vesiculosa

The trap of Aldrovanda vesiculosa, an aquatic insectivorousplant, consists of a pair of lobes (trap-lobes) which bordereach other at the midrib. The central portion of the lobe iscomposed of three cell layers, an inner and outer epidermisenclosing a single middle layer of relatively large cells, whereasthe marginal portion consists only of the two epidermal celllayers. Intracellular potentials of these cells were measuredby the microelectrode technique. All the cells of the lobeswere excitable and had identical membrane potentials at rest( –110 mV) and during action (amplitude, 130 mV). Theaction potential of each cell was elicited by bending a sensoryhair, one of many standing on the inner surface of the centralportion, or by injecting an outward current into another cellin the lobe. Action potentials were propagated throughout thetrap-lobes at a rate of about 8 cm/sec. The maximum rising ratewas 2.7 V/sec and the duration of the action potential was 1sec. (Received August 8, 1981; Accepted October 15, 1981)