The Phototropic Pulvinus of Bean Phaseolus vulgaris L. - Functional Features

Abstract: The laminar pulvinus of bean ( Phaseolus vulgaris L.) is a highly specialized organ for reorienting the lamina, and exhibits positive phototropic curvature. Structural and ultrastructural features of the pulvinus were studied to determine their possible role in its phototropic response. The vascular tissue forms a flexible, relatively inextensible central core enclosed by a starch sheath and surrounded by a multi-layered motor tissue. Phototropic curvature is a result of opposite anisotropic changes in volume of the cells in the exposed and opposite sectors of the motor tissue. Radial inflexibility of the epidermis and axial plasticity constrain these changes to the pulvinar axis. Anisotropic changes in volume of motor cells reduce the osmotic work involved. Motor cells exhibit features that are associated with high synthetic activity: thick cytoplasm with numerous ribosomes, polysomes, RER and SER, well-developed mitochondria and a large nucleus. Numerous, well-developed chloroplasts, with little starch, are increasingly abundant toward the periphery. The intercellular system is limited and partially filled with matrix. Stomata are absent and the motor tissue lacks vascularization. These features support the suggestion that the primary role of the chloroplasts in the photonastic response is photophosphorylation and photosynthetic electron transport (Koller et al., 1995^[339]).     

Blue Light Pulse-Induced Transient Changes of Electric Potential and Turgor Pressure in the Motor Cells of Phaseolus vulgaris L.

Blue light induces both depolarization of membrane potentialin the motor cell and turgor movement in the laminar pulvinusof bean plant. This paper describes the changes of electricpotential and turgor pressure induced in Phaseolus vulgarisL. by blue light pulses. A transient depolarization of membranepotential as large as 40 mV was induced by a short pulse of15 s blue light in motor cells of the laminar pulvinus. Thischange was not an action potential because of the absence ofa refractory period and threshold. The magnitudes of the responsewere dependent on the fluence of light. The response was long-lived,indicating that continuous input of light energy is not requiredfor a sustained response. The potential change was always followedby a transient turgor movement of the pulvinus. A molecular mechanism similar to a model postulated for theblue light response of stomata may operate in the motor cell.However, the direction of the electrical response to blue light(depolarization) in the motor cell was the opposite of thatin the guard cell (hyperpolarization). Turgor change of themotor cell by blue light was also opposite in direction (decrease). (Received February 19, 1988; Accepted June 28, 1988)     

Phototropic Response of the Bean Pulvinus: Movement of Water and Ions

Segmental analysis of the laminar pulvinus of Phaseolus vulgaris L. showed that its phototropic curvature is accompanied by efflux of inorganic ions and water from its contracting sector and a comparable influx into its expanding one. All the major ions, except Na⁺, contributed to this transport, suggesting that the response to light involves changes in the driving force, or conductivity of a wide range of solutes. During the curvature, K⁺ and CI^? made the greatest and equivalent contributions to efflux, but only Cl^? exhibited a matching influx into the expanding sector, while K⁺ influx was much less. Use of the cell pressure probe showed that, as the laminar angle of elevation changed between ?40° to +40°, turgor pressure in the expanding motor cells increased by 0.48 MPa and decreased in the contracting cells by 0.32 MPa. Picoliter osmometry of single-cell samples showed that during this movement vacuolar osmotic pressure remained constant. Thus, changes in turgor pressure resulted from changes in apoplastic, rather than the protoplastic osmotic pressure. Volumetric modulus of elasticity of pulvinar motor cells is very low, showing that their walls are very elastic. These properties increase the effectiveness of converting osmotic work into the large-scale, reversible volume changes responsible for leaf movements.     

Light-driven leaf movements*

Abstract. Mature leaves of many plants re-orientate their laminae photonastically in response to non-directional light signals, and/or phototropically in response to directional light signals, by flexing of pulvini, most commonly subtending their bases. Physiological and structural specializations of the pulvinus enable it to flex, by rapidly undergoing differential and repeatedly reversible axial volume changes (expansion/contraction) in opposite sectors of its motor tissue. Light-driven leaf movements are adaptations that contribute to the efficiency of the photosynthetic apparatus in the leaf. The phototropic response maximizes the harvesting of photosynthetically radiant energy. The photonastic response to dark-to-light transitions increases the interception of light by unfolding the lamina. Another photonastic response modulates the interception of radiant energy by the lamina, allowing it to evade damage by light in excess of its photosynthetic capacity when the leaf is under stress. The same unidentified blue-absorbing pigment system appears to be involved in all these responses. Non-directional light signals are perceived in the pulvinus. Perception of directional light signals may be localized in other parts of the leaf in different plants: for example, the pulvinus in most leguminous species, and the lamina in malvaceous and at least one leguminous species. The perception of non-directional and directional light signals, their transduction to differential volume changes in the target cells, and their transmission between the two where the sites are separate, are discussed.     

Phototropic Response to Vectorial Light in Leaves of Lavatera cretica L

The mechanism by which the mature leaf of certain plants reorients its lamina to face the sun throughout the day was studied in Lavatera cretica L. The photoreceptor for this response differs fundamentally from the one involved in the phototropic growth response, by sensing light as a vector, rather than as a difference in luminous flux. The photoreceptor is located in the veins, which radiate in the plane of the lamina from the pulvinus situated at the junction between the lamina and petiole. The integrated response to the messages from the different veins takes place by differential turgor changes in a motor tissue surrounding the central vascular cylinder of the pulvinus, in which the veins coalesce. The differential turgor in the different segments of the motor tissue determines the orientation of the lamina. The photoreceptor reacts only to a parallel light beam striking the vein obliquely (from above). When half of the lamina is shaded, the leaf does not reorient in response to perpendicular illumination and its reorientation in response to an oblique beam is slower and partial, to a greater extend when the half-leaf is centrifugally illuminated than when it is centripetally illuminated. Application of 2,3,5 tri-iodobenzoic acid to the base of the veins in the shaded half-leaf eliminated all restrictions from the response to centrifugal illumination and totally inhibited the response to centripetal illumination. The results are consistent with a hypothesis that centrifugally illuminated veins generate turgor in their associated motor tissue in the pulvinus by activating K⁺ uptake, while centripetally illuminated veins cause loss of turgor in their associated motor tissue by deactivating K⁺ uptake, which leads to passive leakage of K⁺. When the entire lamina is exposed to oblique illumination, the centrifugally illuminated half and the centripetally illuminated half cooperate in the full response. Shaded parts of the lamina apparently interfere with the response by supplying their associated motor tissue with auxin, which presumably causes in it an active export of protons and concomitant uptake of K⁺, thereby establishing a static “dark turgor” in it.     

Potassium Flux and Leaf Movement in Samanea saman : I. Rhythmic Movement

Samanea leaflets usually open in white light and fold together when darkened, but also open and dose with a circadian rhythm during prolonged darkness. Leaflet movement results from differential changes in the turgor and shape of motor cells on opposite sides of the pulvinus; extensor cells expand during opening and shrink during closure, while flexor cells shrink during opening and expand during closure but change shape more than size. Potassium in both open and closed pulvini is about 0.4 N. Flame photometric and electron microprobe analyses reveal that rhythmic and light-regulated postassium flux is the basis for pulvinar turgor movements. Rhythmic potassium flux during darkness in motor cells in the extensor region involves alternating predominance of inwardly directed ion pumps and leakage outward through diffusion channels, each lasting ca 12 h. White light affects the system by activating outwardly directed K⁺ pumps in motor cells in the flexor region.     

Light-Induced Changes of Electrical Potential in Pulvinar Motor Cells of Phaseolus vulgaris L.

The sealing-off phenomenon of microelectrode tips has oftenbeen observed in plant cells during potential measurements.In motor cells of pulvinus, changes of the sealing-off potentialwere induced by blue light. These light-induced changes of electricalpotential were in the direction opposite to those of the changesin intracellular potential. The results were evaluated and discussedin relation to the changes of the potential difference of themotor cells and turgor movements of the pulvinus. (Received February 2, 1987; Accepted June 29, 1987)     

Light-promoted changes in apoplastic K+ activity in the Samanea saman pulvinus,monitored with liquid membrane microelectrodes

The movement of Samanea saman (Jacq.) Merrill leaflets is a consequence of the re-distribution of K⁺ and anions between motor cells on opposite sides of the pulvinus. We used a K⁺-sensitive microelectrode to study dynamic changes in K⁺ transport through motor-cell membranes during and immediately after change in illumination. Potassium-ion-sensitive and reference microelectrodes were inserted into extensor or flexor tissue of a whole pulvinus in white light (WL). A brief pulse of red light (RL) followed by darkness (D) (a) increased K⁺ activity in the extensor apoplast, indicating K⁺ release by the protoplast; and (b) decreased K⁺ activity in the flexor apoplast, indicating K⁺ uptake by the protoplast. White light after 35–40 min D reversed K⁺ activity in the extensor apoplast to approximately its original value. Blue light substituted partially for WL in this regard. Potassium-ion activity in the flexor apoplast reverted to approximately its original value after 2 h, with or without white illumination. Our data support the hypothesis that K⁺ efflux from extensor cells and K⁺ uptake by flexor cells following a WLRLD transition occurs by way of K⁺ channels.Abbreviations L light - WL white light - RL red light - BL blue light - D darkness     

Interaction of gravi- and phototropic stimulation in the response of maize (Zea mays L.) coleoptiles

The influence of gravitropic stimulation upon blue-light-induced first positive phototropism for stimulations in the same (light source and center of gravity opposite to each other) and in opposing directions was investigated in maize cole-optiles by measuring fluence-response patterns. As a result of gravitropic counterstimulation, phototropic bending was transient with maximum curvature occurring 100 min after stimulation. On a horizontal clinostat, however, the seedlings curved for 20 h. Gravistimulation in the opposite direction acted additively upon blue-light curvature. Gravistimulation in the same direction as phototropic stimulation produced a complex behaviour deviating from simple additivity. This pattern can be explained by a gravitropically mediated sensitization of the phototropic reaction, an optimal dependence of differential growth on the sum of photo-and gravistimulation, and blue-light-induced inhibition of gravitropic curvature at high fluences. These findings indicate that several steps of photo-and gravitransduction are separate. Preirradiation with red light desensitized the system independently of applied gravity-treatment, indicating that the site of red-light interaction is common to both transduction chains.Abbreviations BL blue light - G⁺ stimulation by light and gravity in the same direction (i.e. light source and center of gravity opposite to each other) - G^- stimulation by light and gravity in opposing directions     

Photobiological and Structural Studies of Light-driven Movements in the Solar-tracking Leaf of Lupinus palaestinus Bioss. (Fabaceae)

The compound, palmate lamina of Lupinus palaestinus reorients photonastically, as well as phototropically in response to non-directional and directional light signals, respectively, by structural deformations of pulvini. When the excitation provided by directional light is maintained constant (fluence rate, angle of incidence and azimuth, with respect to the leaflet laminae), the entire lamina reorients towards it at a constant angular velocity over a considerable time interval and displacement. The laminar pulvinules are considerably longer than the subtending common petiolar pulvinus and therefore contribute most to laminar reorientation. The pulvinar region is characterized by transverse folds around its circumference, and longitudinal rib-like thickenings on the external walls of its epidermis that facilitate axial and transverse deformations. Specialized “joints”, at the distal and proximal ends of each pulvinule, contribute most to its flexing. Anthocyanin is notable by its absence. Specialized “motor” tissues surrounding the central vascular core participate in pulvinar deformation by undergoing directional and differential volume changes. The bundle sheath is characterized by numerous starch grains. The multi-layered cortical parenchyma exhibits an abundance of transversely oriented primary pit fields and associated plasmodesmata. When the leaflet lamina rotates around its midrib, the pulvinus twists along its axis, exhibiting epidermal and cortical deformation. The functional significance of these specializations is discussed.     

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)     

Physical strain-mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.     

Morphing structures and signal transduction in Mimosa pudica L. induced by localized thermal stress

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.     

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; )     

Effects of vanadate, N2 and light on the membrane potential of motor cells and the lateral leaflet movements of Desmodium motorium

The lateral leaflets of Desmodium motorium (Houtt.) Merr. exhibit ultradian up- and down movements, which are paralleled by oscillations of the membrane potential of motor cells in the pulvinus. By different treatments we have tested the hypothesis that both that both oscillation-types are causally related. The reactions of the leaflet movement and the membrane potential were evaluated by the following approaches. (1) Application of vanadate. an inhibitor of the proton pump in the plasmalemma. and N₂ suppressed leaflet movements and finally arrested the leaflet in the lower position. Before the oscillations damped out, a strong lengthening in period was found. This indicates that the pump is part of the ultradian clock. A period lenthening and a final suppression of the rhythm by vanadate was also seen in the extracellular electric potential of the pulvinus. Intracellular recordings in situ showed that vanadate application depolarized the motor cells. (2) Light of high fluence rates diminished the amplitude of the oscillations of the membrane potential of single motor cells and shortened the period. The same effects were observed when monitoring the lateral leaflet movement. The leaflet always moved towards the direction of the light. whether it was applied from the abaxial or from the adaxial part of the pulvinus. (3) When light was applied to the pulvinus of lateral leaflets. which had spontancously stopped moving in an upper position. oscillations were induced transiently. This effect was also found for the membrane potential of motor cells in the pulvinus. - Our results thus provide further evidence that the membrane potential controls the volume state of the motor cells in the pulvinus of lateral leaflets of Desmodium motorium .     

Red light enhancement of the phototropic response of etiolated pea stems

In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using ¹⁴C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of ³H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment.     

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     

Control of Paraheliotropism in Two Phaseolus Species

Paraheliotropic (light-avoiding) leaf movements have been associated with high light intensity, high temperature, and drought. We investigated leaf elevation for intact plants, pulvinus bending for excised motor organs, and size change for protoplasts from motor tissue for two Phaseolus species: Phaseolus acutifolius A. Gray, native to hot, arid regions, and Phaseolus vulgaris L., the common bean. Leaf angles above horizontal were measured for central trifoliolate leaflets of intact plants at 24, 27, and 30[deg]C at 500 and 750 [mu]mol photons (400-700 nm) m-2 s-1 over a range of water potentials; equivalent angles were determined for excised motor organs under similar conditions. Diameters were measured for protoplasts from abaxial and adaxial motor tissue over a range of photon flux density values, temperatures, and water potentials. In general, higher photon flux density and temperature resulted in elevation of leaves, bending of excised pulvini, and equivalent changes in protoplast volume (swelling of abaxial protoplasts and shrinking of adaxial protoplasts). In intact plants, lower water potentials yielded greater paraheliotropism; abaxial protoplasts increased in size, whereas adaxial ones did not change. P. acutifolius typically exhibited greater paraheliotropism than did P. vulgaris under the same conditions, a set of physiological responses likely to be highly adaptive in its native arid habitat.     

Induction of beta-glucosidase activity in maize coleoptiles by blue light illumination

The role of beta-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of beta-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by beta-glucosidase, and its degradation compound 6-methoxy-benzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, beta-glucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of beta-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature.     

K+ Gradients in the Pulvinus of Phaseolus coccineus during Leaf Movement*

We used quantitative histochemistry to investigate the tissue-specific compartmentation of potassium in the laminar pulvinus of Phaseolus coccineus L. at day and night positions of diurnal leaf movement. The assay was based on the potassium-dependent activation of pyruvate kinase. Total potassium levels of pulvini were higher in the light than in the dark [0.88 and 0.57mol (kg dry weight)^?1, respectively]. Transverse compartmentation of potassium was studied on three tissue slices, representing the middle part, petiolar and laminar sides of individual pulvini. These were dissected further into 10 distinct subsamples (bundle; motor tissues: extensor, flexor; flanks). In the day position the amount of potassium in the extensor was higher than in the night position [1.92 and 1.50 mol (kg dry weight)^?1, respectively]. Flexor changes were opposite [1.13 and 1.65 mol (kg dry weight)^?1, respectively]. In the day position there was a steep and consistent increase in potassium content from the innermost to the outermost zones of the extensor. In the night position this was much more variable. Comparable gradients were not detected in flexor samples. Here highest amounts of potassium were recovered from the middle of the motor tissue. The data specify distinct tissue regions involved in osmotic adjustment during leaf movement in Phaseolus coccineus.