ABSTRACILeaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K+, Cl and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K+ currents in extensor and flexor cells that increase slowly (t½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K+ currents is adequate to account quantitatively for K+ loss, previously measured in vivo during cell shrinkage. The K+ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light-and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.Leaflet movements in nyctinastic (night closure) plants often involve significant changes in the volume and up to severalfold variation in the ionic content of motor cells in the pulvinus (reviewed in Ref. 21). These variations may occur in response to light, darkness, and an endogenous biological clock. Cells in the extensor region of the pulvinus take up K+ and Cl-as they swell during leaflet opening, and lose both ions as they shrink during leaflet closure, while cells in the opposing (flexor) region behave in the reverse manner (12,22,23,25,30,32).We undertook this study to examine a possible role for K+ channels in leaflet movement-related K+ fluxes and changes in cell volume in the nyctinastic legume Samanea saman. K+ channels have already been described in giant algae (1, 5) and in protoplasts isolated from wheat mesophyll cells (13,14), Vicia faba guard cells (27,29) Protoplast Isolation. Terminal secondary pulvini from the fourth to ninth leaf (counting from the apex) were harvested 2 to 3 h after the beginning of the light period in the growth chamber, or 2 to 3 h after sunrise in the greenhouse. Protoplasts were prepared by enzymic digestion of slices of extensor or flexor tissue (pooled separately), as described in (7) but with the following modifications. (a) The osmotic pressure of the pre-digestion solution was raised in two steps to 680 mosmol to ensure plasmolysis. (b) Pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan) was added to the digestion solution (which contained cellulase, pectinase and Driselase) to a final concentration of 0.2% (w/v). (c) A second purification step was added, as follows: the cells collected from the Ficoll interface were layered again on a sucrose gradient (2), spun at 60 to IOOg for 5 min, and collected and kept on ice for up to 24 h for patch-clamp experiments.Forty to fifty protoplasts of each type, flexor and ...
The patch-clamp technique was used to study passive movements of ions through the plasmalemma of wheat leaf protoplasts. This method overcomes the problems inherent in conventional electrophysiological study of plant cells. Changes in conductance were recorded in patches excised from the plasmalemma. Two types of patches were observed: (i) regions of low channel density, where discrete single-channel currents could be resolved and conductance ranged from 10 to 200 picosiemens and (ii) regions of high channel density, where single-channel currents could not be resolved and conductance was on the order of a few nanosiemens. The results indicate a striking similarity between animal and plant cell membranes in the basic phenomena of transport. Moreover, the approach used constitutes a new degree of refinement in the study of processes of regulation, pathology, and toxicity in plants.
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