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     

Ultradian Rhythms in Desmodium

The leaves of Desmodium gyrans (L.F.) DC show circadian movements in the terminal and ultradian movements of the lateral leaflets. The movements are due to swelling and shrinking of motor cells in special organs. The anatomy of these pulvini is described for the lateral leaflets. Data from electrophysiological recordings using microelectrodes inserted into the lateral pulvini, together with treatments that affect the proton pumps and ion channels, have been used to develop a physiological model of the ultradian leaflet movement. It explains the oscillations in the motor cells as being due to a change between a pump state and depolarization. During the pump state, ions are taken up, causing water influx and swelling of the motor cells. Depolarization causes loss of ions and water efflux (the motor cells shrink). The roles of calcium and the phosphatidyl inositol signal chain are discussed on the basis of experiments using chemical agents that affect these processes. Since calcium oscillations are known to occur in organisms in both time and space, an attempt has been made to simulate the situation in Desmodium pulvini by a model of specially coupled oscillators. Effects of different other treatments of the lateral pulvini are discussed. Oscillations in the minute range seem to be more common and some might be related to turgor regulation and ion uptake comparable to the situation in Desmodium. The ultradian control of the lateral pulvini and the circadian control of the terminal pulvini are apparently based on different mechanisms.     

Oscillations of apoplasmic K+ and H+ activities in Desmodium motorium (Houtt.) Merril. pulvini in relation to the membrane potential of motor cells and leaflet movements

The lateral leaflets of Desmodium motorium exhibit rhythmic upward and downward movements with a period in the minute range. Apoplasmic K⁺ and H⁺ activities were monitored in situ in the abaxial part of the pulvini with ion-selective microelectrodes. An extracellular electric potential was recorded simultaneously. The apoplasmic H⁺ activity of all pulvini exhibiting a regular rhythm of the extracellular electric potential oscillated with the same period between about 10 and 20 mM. The apoplasmic K⁺ activity was high when the membrane potential of the motor cells was depolarized (about 36 mV) and the cells were shrunken. In contrast, the apoplasmic K⁺ activity was low in the swollen state of the motor cells, when the membrane potential was hyperpolarized (about -136 mV). The volatile anesthetic enflurane suppressed reversibly the movement of the leaflets. The same treatment also arrested spontaneous oscillations in the apoplasmic K⁺ activity in the pulvinus. The apoplasmic K⁺ activity oscillated roughly in phase with the K⁺ activity between pH 6.6 and 6.0. Application of white light disturbed the rhythm and increased the extracellular pH. Our results indicate that the physiological mechanism that drives the lateral leaflet movements of Desmodium motorium is closely related to the osmotic motors mediating the leaf movements of Mimosa, Samanea and Phaseolus.Abbreviations E_m membrane potential - E_ex extracellular electric potential - H_ex extracellular H⁺ activity - K_ex extracellular K⁺ activity - R_ex extracellular electrical resistance B. Antkowiak was supported by the Stiftung Volkswagenwerk.     

Effects of light on ultradian rhythms in the lateral leaflets of<Emphasis Type="Italic">Desmodium gyrans</Emphasis>

Rhythmic up-down movements were studied in the lateral leaflets ofDesmodium gyrans (L.F.) DC. These were recorded with a video-computer system, whereby the digital video signals from a CCD camera were processed with special software. Under control conditions (24°C and 0.1 nM cm^-2 s^-1 of stable, dim light), the average period of lateral leaflet movement was 3.5 min. In the presence of light stimuli (for 2 min), those leaflets always moved toward the light, regardless of where it was applied to any axial part of the pulvinus. The strongest effect was manifested by a reduced amplitude of movement and, thus, a shortened period, which could be up to ~43% less under moderate light intensity (5 nM cm^-2 s^-2). Oscillations regained their original regularity over ~10 cycles after the light stimulus was removed. In addition, these oscillations temporarily disappeared after long exposure (~10 min) under moderate light, or when the leaflets were quickly exposed to a higher intensity (~12 nM cm^-2s^-1). Therefore, we have now demonstrated that light can affect physiological parameters that are involved in the control of oscillations.     

Light-dependent changes in the leaflet movement rhythm of the plant Desmodium gyrans

The movements of the lateral leaflets of the Indian telegraph plant Desmodium gyrans (L. F.) DC, have earlier been studied in detail with regards to the effects of chemicals, DC currents, and static magnetic fields. In the present paper we have discussed the oscillation of the lateral leaflets under the influence of white light of various light levels (0-75 micromol x m(-2) x s(-1)), produced by an array of LEDs (light emitting diodes). LEDs were used in contrast to fluorescense tubes as in earlier studies in order to minimize changes of wavelength when light intensity was decreased or increased. Furthermore, care was taken to ensure that the temperature in the experimental chamber was constant. When the oscillations were first monitored in bright light, the oscillations were found to be very rapid and with decreasing light intensity the oscillations slowed down. For light levels lower than about 20 micromol x m(-2) x s(-1) the period of the oscillation of the lateral leaflets was almost constant (or even decreased slightly towards complete darkness). We also show that the oscillations could completely stop under prolonged darkness (for longer than about 6 h) and that such halted oscillations could be restarted in most of the leaflets when he light was turned back on. Such stopping of the oscillation of the lateral leaflets in prolonged darkness suggests that these short period oscillations of the lateral leaflets could have a daily component and in natural environment these oscillations could serve the purpose of optimising the amount of light falling on the leaflets or/and facilitating transpiration of water through stomata. Such a finding could have an implication for the answer to the long standing question of adaptive significance of short period oscillation of the Indian telegraph plant Desmodium gyrans (L. F.) DC.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

HELIOTROPIC LEAF MOVEMENT RESPONSE TO H+/ATPase activation,H+/ATPase inhibition,AND K+ CHANNEL INHIBITION IN VIVO

The pulvinus, located at the base of soybean leaflets, is both the light perception and motor organ for heliotropic leaf movements. Our objective was to investigate the role of plasma membrane H⁺/ATPase and TEA-sensitive K⁺ channels in mediating pulvinar response to light. The plasma membrane H+/ATPase activator, fusicoccin, plasma membrane H⁺/ATPase inhibitors, vanadate and erythrosin-B, and the K⁺ channel blocker TEA were introduced to the intact pulvinus through the transpiration stream. The pulvinus was illuminated by a vertical light beam of 1,400 μmol m^-2 s^-1 to stimulate leaf movement. Leaf orientation was measured every 5 min for 60 min of illumination. All compounds tested inhibited pulvinar bending, but concentration and uptake time required for inhibition varied: 12.5 μM fusicoccin reduced leaf movement after 3 hr uptake, 2 mM vanadate reduced leaf movement after 6 hr uptake, 100 μM erythrosin-B reduced leaf movement after 3 hr uptake, and 15 mM TEA reduced leaf movement after 6 hr uptake. In all cases final leaf angle was reduced by higher concentrations and/or increased time for uptake of the chemical into the pulvinus. Results support the hypothesis that the proximal mechanism of heliotropic movement is similar to that of nyctinastic movements.     

Short period leaf movements in Oxalis regnellii

Oxalis regnellii Mig. is a trifoliate plant, and the three leaflets usually show synchronized up and down movements with a circadian period of 26–27 h. The three leaflets can also perform desynchronized ultradian oscillations, and we report on such rhythms under different conditions. A study of the occurrence of ultradian leaf movement rhythms as a function of irradiance is presented. At an irradiance of approximately 1 μW cm⁻², the occurrence was maximal and ca 30%. The periods varied from 5 to 15 h. Four other cases of ultradian rhythms in different conditions are also presented. In one case spontaneous ultradian rhythms occurred, and in another, two of the leaflets showed ultradian rhythms when the third leaflet had received a light pulse. In two more cases, the three leaflets on a leaf were separated by physical cuts along the petiole between the pulvini; in both cases the period was approximately 5 h. Possible mechanisms to explain the ultradian rhythms in Oxalis regnelli are discussed.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate,and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (～3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10–15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Substances interfering with phosphatidyl inositol signalling pathway affect ultradian rhythm ofDesmodium motorium

The ultradian rhythm of the lateral leaflets ofDesmodium motorium}(Houtt.) Merril. was recorded with a picture analysis method using a video camera and a computer. The periods are in the minute range and depend strongly on temperature. The phosphatidyl inositol signal chain might be involved in the ultradian rhythm of the lateral leaflet movement of Desmodium motorium:Myoinositol shortens the period length and reduces the known period lengthening effect of lithium ions. Neomycin, which inhibits the hydrolysis of phosphatidylinositol-4,5 -biphosphate to inositol-4-phosphate and diacylglycerin, lengthens the period of the rhythm at low concentrations (0.2 mM). Higher concentrations shorten the period, perhaps by activating G protein. Mastoparan, which activates G protein, shortens period likewise. The G protein agonists fluorid ion and ethanol are toxic for the lateral leaflets and could therefore not be used to test the involvement of G protein. The intracellular Ca ²⁺ antagonist 3,4,5-trinietlioxybeiizoic acid 8-(diethylamino)octylester lengthens the period of the rhythm. This indicates, that release of Cas ²⁺ from intracellular stores is important for the lateral leaflet movement rhythm.     

Phase response curve for the ultradian rhythm of the lateral leaflets of Desmodium gyrans using DC current pulses

In the present study the leaf movement rhythm was perturbed by the application of DC current pulses (15 microA, 10 seconds, voltage applied: 10 V) to the upper part of the pulvinus, passing through the pulvinus and its stalk. The pulses were applied at four different positions of the leaflets: when the leaves were at the lowermost position, when moving up, at the uppermost position and when moving down. The pre-perturbed and the post-perturbed rhythms were compared. We found that the rhythms were shifted in phase and the phase shifts observed at the four different positions of the leaflets were significantly different in magnitude as well as direction. Furthermore, we could also observe phase advances, which is in contrast to an earlier finding. A phase response curve (PRC) was constructed to illustrate the sensitivity of the oscillating leaflet system to DC pulses. Substantial delays of about 50 s (as compared to the period of about 200 s) were obtained when pulses were administered at the lowermost position and when leaflet were moving upwards, while advances or no phase shifts were recorded in the uppermost position and when leaflet were moving down respectively.     

Dynamic aspects of the response of the pulvinus in the leaf of bean plants(Phaseolus vulgaris L.) to photoexcitation

The pulvinus of bean (Phaseolus vulgaris L) responds to unilateral photo-excitation by phototropic curvature. Osmotically active solutes and water are transported from its exposed to the opposite sector of its motor tissue, resulting in differential changes in turgor pressure in these sectors and generation of a trans-pulvinar torque. A null-point approach was used to non-invasively study these dynamic changes in the terminal leaflet of bean. A variable torque was applied perpendicular to the midrib, to restrain laminar movement by precisely and continuously counteracting the generated torque. This equilibrium prevented curvature of the pulvinus and the associated opposite axial changes in volume in the opposite sectors of its motor tissue. The laminar torques measured were used to estimate stresses (changes in turgor pressure) generated within the motor tissue. These stress values were used to derive the corresponding changes in osmotic pressures and in solute concentration. Skotonastically downfolded leaflets were excited with white light to study their combined dynamic response to photonastic and phototropic excitation. Photonastically unfolded (horizontal) leaflets were excited with blue and red light, alone and in combination, to determine the spectral dependence of the dynamic pulvinar responses tophototropic excitation by itself.     

Phytochrome and Circadian Clocks in Samanea: Rhythmic Redistribution of Potassium and Chloride within the Pulvinus during Long Dark Periods

Previous investigations with the electron microprobe reveal that the movements of Samanea leaflets are correlated with massive redistribution of K within the pulvinus. Evidence is now presented that Cl moves with K, whether plants are in white light or darkness, whether or not the amplitude of free running oscillations has damped, and whether or not the rhythm has been rephased by phytochrome photoconversion. The mid-extensor to mid-flexor ratio of K + Cl is correlated with leaflet angle under all conditions. Total Cl in both inner cortex and motor region is approximately 0.6 as high as K. The stoichiometry between Cl and the migratory fraction of K is close to, but not precisely 1:1 in all regions of the pulvinus, suggesting that other ions or systems may also be involved in the balancing of electrical charges.     

An Experimental Analysis and Comparison of Three Rhythms of Movements in Bean (Phaseolus Vulgaris L.)

Three types of rhythmic movements of Phaseolus vulgaris L. (pole beans) were examined collectively and their characteristics compared. Although the ultradian rhythms of shoot circumnutation and leaf movement, as well as the circadian rhythm of leaf movement, occurred simultaneously, each rhythm could be expressed independently of the other two. Shoot circumnutation and ultradian leaf movements displayed the same period (80 min at 25°C and Q₁₀⋍2), while the period of the circadian leaf movements was not temperature dependent (Q₁₀⋍1). Interaction into the plant between two ultradian rhythms (shoot circumnutation and ultradian leaf movement) with the same period and coexistence in the pulvinus of an ultradian with a circadian rhythm are discussed.     

A method to record circadian plant movements, with application to Oxalis leaf rhythms

A transducer was developed to record the circadian movement of the individual leaflets in Oxalis regnellii Mig. The method can easily be adapted to measure other kinds of plant movements as well. It is based on the detection of the shadow each leaflet casts on the small side of a specially formed Perspex plate. The light is guided through the Perspex and collected by a phototransistor, which provides an electrical signal that is proportional to the light intensity falling onto it. The output signal can be made a linear function of the leaf angle. This equipment was used in experiments to study the coupling between the 3 leaflets in Oxalis . Pulses of 4 h of red light were given to one of the leaflets, the two others were shielded from the light. A phase response curve was determined for each leaflet, but there was no significant difference in the phase response between the 3 leaflets. Experiments were also made in which the 3 leaflets were separated physically by cuts along the petiole between the pulvini. In this case ultradian oscillations were observed.     

Rhythmic and Blue Light-Induced Turgor Movements and Electrical Potential in the Laminar Pulvinus of Phaseolus vulgaris L.

The well-known circadian movement in Phaseolus leaves is usuallysuperimposed with small and somewhat irregular rhythmic changesof pulvinar movement. This study examines the relationship betweenthese small pulvinar movements and the membrane potential ofthe motor cell as well as the effect of light on them. Bluelight affected both the movements and potential while red andgreen lights of the same photon flux had little effect. Also,the difference in the membrane potential between the extensorand flexor cells was found to be closely related to the rhythmicturgor movement of the pulvinus. Changes in the potential differencealways preceded the movement. Sequential changes of the potentialdifference and turgor pressure in the motor cells, includingthe light-induced ones, are discussed in relation to the leafmovement. (Received August 8, 1985; Accepted November 11, 1985)     

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)     

Nutations in Shoots and in Desmodium Lateral Leaflets,Nyctinastism and Seismonastism in Mimosa pudica. Comparison and Evolution of Morphology and Mechanism

Four plant movements are considered: circumnutation of apeces of Phaseolus, nutations (ultradian movement) of lateral leaflets of Desmodium gyrans, nyctinastim and Mimosa pudica seismonastic movement. The rhythms mechanisms are briefly summarized except for seismonastism: in this case, experimental evidence progressively accumulated, at first indirect and recently direct is described, leading eventually to conclusions about the existence and function of contractile proteins. Indeed, in spite of the nowadays clear evidence, the mechanism has not yet gained total recognition among plant physiologists and it is usually overseen or ignored in a reductionist approach. From these four exemplative cases, an interesting evolutionary organization change is apparent in movement, morphogenesis, chemistry and mechanism.     

Site of light perception and motor cells in a sun-tracking lupine (Lupinus succulentus)

From the results of shading experiments and irradiation of leaves from different directions, the pulvinus has been identified as the site of light perception in leaves of the sun-tracker Lupinus succulentus Dougl. Within the pulvinus, the light-sensitive region is located on the adaxial surface of the base of each leaflet. This is coincident with the site of the motor cells which are located in the peripheral four cell layers. Motor cells were visualized by microscopy and undergo dramatic changes in cell volume as leaflets incline to orient perpendicular to an oblique beam of light. Communication between leaflets appears to be minimal because each leaflet has the capacity to perceive and orient towards a light beam independently of the others. Hypotheses for the mechanism of directional light perception are discussed.     

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.