Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini

Abstract

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide. White followed by darkness (7,8). Similarly, during circadianrhythmic leaflet movement, K+, Cl-, and water are taken up alternately and rhythmically by flexor and extensor cells (2).The plasma membrane H+-ATPase of plant cells has a primary role in coupling ATP hydrolysis to solute movement (22). Outwardly directed H+ transport by the H+-ATPase produces both a membrane electrical potential and a pH gradient. The membrane electrical potential drives the uptake of cations, mainly K+, whereas the pH gradient promotes the uptake of anions such as Cl-and neutral molecules such as sucrose through H+-coupled cotransport. For example, during blue light-induced opening of leaf stomata, H+ extrusion from guard cells creates an electrical gradient that drives K+ uptake through K+-selective ion channels (1,18,19); stomatal opening is inhibited by the H+-ATPase inhibitor vanadate (21). Irradiation of isolated S. saman pulvini also causes H+ fluxes, membrane potential changes, and K+ movement, which regulate pulvinar bending and, hence, mediate light-induced leaflet movement (7,8). Similarly, opposing changes observed in apoplastic H+ and K+ concentrations in the extensor region of the Phaseolus pulvinus also support a role for the H+-ATPase in K+ uptake during rhythmic leaflet movement (24). Potassium channels have been described in cells involved in turgor-mediated movements. Voltage-gated K+ channels activated by membrane depolarization have been observed in patch-clamp studies of plasma membranes of S. saman protoplasts (9) and of Vicia faba guard cell protoplasts (19,20). These channels might serve as the pathway for K+ efflux from shrinking cells. Hyperpolarization-activated K+ channels have been reported in guard cell protoplasts of V. faba (20) and in S. saman protoplasts (9). These channels might serve as a pathway for K+ influx into swelling cells.We are studying the transduction pathway mediating lightinduced changes in K+ flux. We have examined the effect of light on K+ channels by measuring the relative membrane potential of S. saman protoplasts exposed to light and the sensitivity of this potential to external K+. We present evidence here that white and blue light induce K+ channel opening in the plasma membranes of extensor protoplasts while inducing K+ channel closure in the plasma membranes of flexor protoplasts.

Signal Transduction in Leaf Movement

“…In the dark period, the closed inward-directed Kt channels of extensor cells are opened within 3 min by blue light, as demonstrated by a K+-induced depolarization reported by the added fluorescent dye (Kim et al, 1992). Conversely, the channels of flexor cells, open in the darkness, are closed by blue light (Kim et al, 1992). In the light period, however, the situation is more complex (Kim et al, 1993).…”

“…Isolated pulvinar protoplasts are also responsive to light signals (Kim et al, 1992(Kim et al, , 1993. In the dark period, the closed inward-directed Kt channels of extensor cells are opened within 3 min by blue light, as demonstrated by a K+-induced depolarization reported by the added fluorescent dye (Kim et al, 1992).…”

“…They monitored the state of these K + channels, using a membrane-permeable dye sensitive to membrane potential to report depolarization produced by added K + ions entering the protoplasts through inward-directed K + channels. Although this method monitors the channels indirectly, these authors used a combination of studies of K + concentration dependence, ionic specificity, and inhibitor sensitivity to show that the dye was, in fact, responding to a depolarization dependent on inward-directed K + channels (Kim et al, 1992). Kim et al (1992) found that the inward-directed K + channels were open in flexor protoplasts and closed in extensor protoplasts during the normal dark period.…”

“…Although this method monitors the channels indirectly, these authors used a combination of studies of K + concentration dependence, ionic specificity, and inhibitor sensitivity to show that the dye was, in fact, responding to a depolarization dependent on inward-directed K + channels (Kim et al, 1992). Kim et al (1992) found that the inward-directed K + channels were open in flexor protoplasts and closed in extensor protoplasts during the normal dark period. This is consistent with the expected behavior of these cells in the intact plant; during the dark period flexor cells are swollen from the uptake of K + and water and extensor cells are shrunken, having lost K + and water.…”

Dance of plants with circadian clock

“…K ϩ and main counterion to K ϩ , Cl Ϫ , increase in the swelling cells and simultaneously decrease in the shrinking cells (Satter and Galston 1981;Satter et al 1988). The K ϩ permeability is controlled by light (Lowen and Satter 1989, Kim et al 1992Suh et al 2000) and the circadian clock (Kim et al 1993). The extensor and flexor cells in S. samae have four genes encoding K ϩ channels (Moshelion et al 2002a).…”