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)     

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.     

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     

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; )     

STRUCTURAL SPECIALIZATION OF THE SITE OF RESPONSE TO VECTORIAL PHOTO-EXCITATION IN THE SOLAR-TRACKING LEAF OF LAVATERA CRETICA

Structural features of the pulvinus of the solar-tracking leaf of Lavatera cretica L. that are involved in its capacity for omnidirectional and fully reversible bending in response to vectorial excitation of the lamina were studied by light- and scanning electron microscopy. Pulvini that had bent in the plane at right angles to the midvein were bisected along that plane and the symmetrical tissues of the expanded and contracted flanks were compared. The pulvinus contains a central vascular core and exhibits a transversely furrowed exterior. These specialized features enable the fully mature tissue of this region of the petiole to bend reversibly. The epidermis, chlorenchyma, peripheral collenchyma, and cortical parenchyma in the pulvinus form concentric, radially symmetric sheaths around the vascular core and exhibit structural features in their cell walls that would allow considerable changes in cell volume and consequently enable the omnidirectional bending of this pulvinus. Thickened wall portions of the pulvinar epidermis and peripheral collenchyma exhibit a highly specialized architecture, consisting of alternating thick and thin strips, that enhances their flexibility, while maintaining mechanical support. Cell walls of the chlorenchyma and the cortical parenchyma are thin and capable of reversible infolding. Those of the cortical parenchyma also exhibit numerous prominent transverse pit fields, indicative of anisotropic orientation of their microfibrillar lattice transverse to the pulvinar axis. This orientation is compatible with elastic deformation of the cortical parenchyma cells along the pulvinar axis. Filament-like cytoplasmic strands were observed along the walls of pulvinar motor cells, predominantly transverse to the pulvinar axis, but their function (if any) in volume changes of these cells is unknown.     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

The shortening and action potential of the cortex in the main pulvinus ofMimosa pudica

The shortening and action potential of the upper and lower cortices of the main pulvinus ofMimosa pudica were recorded simultaneously. The shortening and action potential were observed only in the lower cortex. The extensibility and the excitability of the cortex are discussed.     

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)     

Age-dependent changes of the ion content and the circadian leaf movement period in thePhaseolus pulvinus

The circadian movement of the lamina of primary leaves ofPhaseolus coccineus L. depends on circadian changes of the K⁺, Cl^- and (depending on the Cl^- availability) malate content in the swelling and shrinking motor cells of the laminar pulvinus. After sowing in soil, the laminar pulvinus develops within about 26 days. When the leaves emerge from the soil (about 6 days after sowing) and the pulvinus starts with the diurnal movement (about 9 days after sowing) the pulvinar dimensions are about half of those of the mature pulvinus. The anatomical structure, however, is basically the same as in the developed pulvinus. In soil-grown plants, the K⁺, Cl^- and malate content as well as the period length of the circadian leaf movement rhythm change in the developing pulvinus. In the embryo of the dry seed, the Cl^- content is low (0.03 mmol g^-1 DW), the K⁺ content, however, 22-fold higher than the Cl^- content. When the leaves emerge from the soil, the pulvinar K⁺ and Cl^- content is the same as in the whole embryo of the dry seed. In the developing pulvinus the K⁺ content increases by a factor of 2 and the Cl^- content by a factor of 41 in the mature pulvinus. The pulvinar malate content increases between the 6th and 10th day from about 40 to 180Μmol g^-1 DW, then decreases until the 17th day and remains thereafter on a low level (around 80 Μmol g^-1 DW). These results indicate that the Cl^- availability increases in the developing pulvinus with age. It explains furthermore why in young leaves malate was found as counterion to K⁺ in the osmotic leaf movement motor, in older ones, however, Cl^-. The circadian leaf movement starts 9 days after sowing. The period length decreases during the development of the pulvinus from 31.3 to 28.6 h in leaves of intact soil-grown plants. In leaves which were cut from the plants and immersed with their petioles in distilled water, the age dependent decrease of the period length is also found. However, the period lengths are shorter by more than 1 h than in the leaves of intact plants. The increasing Cl^- availability in the developing pulvinus does not seem to be the cause for the age dependent shortening of the period length, because the period length in 22 days old Cl^- deprived pulvini is the same as in 22 days old pulvini with a high Cl^- content.     

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; )     

Changes in mitogen-activated protein kinase activity occur in the maize pulvinus in response to gravistimulation and are important for the bending response

The maize (Zea mays L.) pulvinus was used as a model system to study the signalling events that lead to differential growth in response to gravistimulation in plants. The pulvinus functions to return tipped plants to vertical via differential elongation of the cells on its lower side. By performing immunokinase assays using total soluble protein extracts and an antibody against mammalian ERK1, a mitogen‐activated protein kinase (MAPK)‐like activity was assayed in pulvini halves harvested at various time points after tipping. We detected a reproducible alternation of higher levels of activity occurring between the upper and lower halves of the pulvinus between 75 and 180 min after tipping, with a sustained increase in the upper half occurring at the end of the time‐course. This timing roughly corresponds to the presentation time for maize (i.e. the amount of time that the plant needs to be tipped before it is committed to bend), which occurs between 2 and 4 h. Treatment of maize stem explants with an inhibitor of MAPK activation, U0126, led to a reduction in the activity of this kinase, as well as an almost 65% reduction in bending as measured at 20 h. Rinsing out of the inhibitor resulted in recovery of both bending and kinase activity. It is possible that changes in MAPK activity in the gravistimulated pulvinus are part of a signalling cascade that may help to distinguish between minor perturbations in plant orientation and more significant and long‐term changes, and may also help to determine the direction of bending.     

Movement of water in conjunction with plant movement visualized by NMR imaging

The water distribution in the pulvinus of Mimosa can be visualized by an NMR imaging technique. After stimulation of a Mimosa plant, water in the lower half of the main pulvinus disappeared, the water previously contained in this area seeming to be transferred to the upper half of the main pulvinus. Movement of the water in conjunction with Mimosa movement was visualized sequentially by a non-invasive NMR imaging procedure.     

Chloride ion efflux during an action potential in the main pulvinus ofMimosa pudica

The shortening, action potential and Cl⁻-efflux of the excised lower half cortex in the main pulvinus ofMimosa pudica were simultaneously recorded. The mean values±(S.E.) for Cl⁻-efflux and shortening were 183±18 picomoles/mg fresh weight/impulse and 87.0±2.2 μm, respectively.     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium.Key words: actin, calcium, pulvinus movement, the tannin vacuole, Mimosa pudica     

Transport of the Auxin 2,4-Dichlorophenoxyacetic Acid Through Absiccion Zones, Pulvini, and Petioles of Phaseolus vulgaris

Measurements were made of the transport of 2,4-dichlorophenoxyacetic acid-¹⁴C (2,4-D) through segments cut from the region of the distal abscission zone in young and old primary leaves of Phaseolus vulgaris L. When old leaves were used basipetal transport of 2,4-D in segments including pulvinar tissue, abscission zone, and petiolar tissue was much less than in wholly petiolar segments. In both young and old plants, segments consisting entirely of pulvinar tissue transported 2,4-D basipetally at a velocity about half that in petiolar tissue. At both ages the flux of 2,4-D through pulvinar tissue was less than that through petiolar tissue. In segments from old leaves the flux through pulvinar tissue was much less than in young plants; the flux through petiolar tissue changed little with age. There was no change with age in the velocity of basipetal transport. The distribution of ¹⁴C along segments including the abscission zone showed no marked discontinuity. It was concluded that the pulvinus limited the basipetal movement of 2,4-D through segments from old leaves which included both pulvinar and petiolar tissue, but there was no evidence that the abscission zone itself was a barrier to auxin transport.     

Rapid plant movements triggered by action potentials

Rapid bendings of the pulvinus inMimosa pudica, of the trap lobes inDionaea muscipula andAldrovanda vesiculosa, and of the tentacle in Drosera are triggered by action potentials in their motor cells. The action potential ofMimosa may be a C1⁻-spike, and that ofDionaea andAldrovanda may be a Ca²⁺-spike. Propagation of action potentials in the petiole or motor organ is thought to be electrotonie, cell-to-cell, transmission. The Ca 2+ release from unidentified organelles in the pulvinus or the Ca²⁺ influx of the cells in the trap with the action current and activation of contractile fibrillar network having ATPase activity in the cytoplasm must be involved in the rapid bending. Contractions of fibrils may open pores in the membrane of the motor cells upon activation. Outward bulk flow of the vacuolar sap through these pores, due to the pressure inside the cell, must result in turgor loss of the motor cells and then the bending of the organ.     

IAA-induced hyperpolarization of the membrane potential in isolated cells fromMimosa pulvinus

Upon treatment with 10⁻⁴ M IAA the membrane potential of an isolated cell from the main pulvinus, ofMimosa pudica L. depolarized by about 6 mV in 2–5 min, but later it gradually hyperpolarized by about 30 mV. The membrane potential of a motor cell in the main pulvinar tissue hyperpolarized by about 80 mV 1 hr after application of 10⁻⁴ M IAA.     

Pulvinus activity,leaf movement and leaf water-use efficiency of bush bean (<Emphasis Type="Italic">Phaseplus vulgaris</Emphasis> L.) in a hot environment

Pulvinus activity of Phaseolus species in response to environmental stimuli plays an essential role in heliotropic leaf movement. The aims of this study were to monitor the continuous daily pulvinus movement and pulvinus temperature, and to evaluate the effects of leaf movements, on a hot day, on instantaneous leaf water-use efficiency (WUE_i), leaf gas exchange, and leaf temperature. Potted plants of Phaseolus vulgaris L. var. Provider were grown in Chicot sandy loam soil under well-watered conditions in a greenhouse. When the second trifoliate leaf was completely extended, one plant was selected to measure pulvinus movement using a beta-ray gauging (BRG) meter with a point source of thallium-204 (²⁰⁴Tl). Leaf gas exchange measurements took place on similar leaflets of three plants at an air temperature interval of 33–42°C by a steady-state LI-6200 photosynthesis system. A copper-constantan thermocouple was used to monitor pulvinus temperature. Pulvinus bending followed the daily diurnal rhythm. Significant correlations were found between the leaf-incident angle and the stomatal conductance (R ² = 0.54; P < 0.01), and photosynthesis rate (R ² = 0.84; P < 0.01). With a reduction in leaf-incidence angle and increase in air temperature, WUE_i was reduced. During the measurements, leaf temperature remained below air temperature and was a significant function of air temperature (r = 0.92; P < 0.01). In conclusion, pulvinus bending followed both light intensity and air temperature and influenced leaf gas exchange.     

Blue-light-dependent osmoregulation in protoplasts of Phaseolus vulgaris Pulvini.

Blue light was found to induce shrinkage of the protoplasts isolated from first-leaf lamina pulvini of 18-day-old Phaseolus vulgaris. The response was transient following pulse stimulation, while it was sustainable during continuous stimulation. No apparent difference was found between flexor and extensor protoplasts. Protoplasts of the petiolar segment located close to the pulvinus showed no detectable response. In the plants used, the pulvinus was fully matured and the petiole was ceasing its elongation growth. When younger, 12-day-old, plants were used, however, the petiolar protoplasts did respond to blue light. The pulse-induced response was similar to that in pulvinar protoplasts, although the response to continuous stimulation was transient and differed from that in pulvinar protoplasts. No shrinkage was induced in pulvinar protoplasts when the far-red-light-absorbing form of phytochrome was absent for a period before blue-light stimulation, indicating that the blue-light responsiveness is strictly controlled by phytochrome. Inhibitors of anion channels and H(+)-ATPase abolished the shrinking response, supporting the view that protoplasts shrink by extruding ions. The response of pulvinar protoplasts is probably involved in the blue-light-induced, turgor-based movement of pulvini. The blue-light responding system in pulvini is suggested to have evolved from that functioning in other growing organs.