Potassium Flux and Leaf Movement in Samanea saman

Concentrations of K, Cl, P, S, and Ca in extensor and flexor protoplasts from open pulvini of the nyctinastic tree Samanea saman were estimated using x-ray microanalysis. This technique is particularly suitable when absolute bumbers of protoplasts are low, because less than 100 protoplasts are required to obtain statistically significant data. Flexor protoplasts contain similar concentrations of P and S but almost twice as much K and Cl as extensor protoplasts. Low (2). Although sorbitol was the major external osmoticum throughout protoplast isolation, it is not stable in the electron beam, and thus it had to be replaced before mounting the protoplasts for x-ray analysis. We used a final wash of 60 mm sucrose with 280 mm NaCl, RbCl, NaBr, or RbBr to replace the sorbitol (normally 500 mM) while maintaining approximately the same osmotic concentration.' Supported by grants from the National Science Foundation to R. L. S.2 Samanea saman has been reassigned to the genus Pithecolobium. We retain the name Samanea for continuity with earlier literature.3 Because x-ray analysis detects total elemental content without regard to ionic form, Na, Cl, K, Mg, P, S, Rb, and Br are used to designate these elements, even though they are generally present in ionized form.This step was performed at 0°C to minimize uptake of ions from the wash solution. Washes with LiNO3 or organic anions were considered because their low atomic weight components would interfere less with x-ray analysis, but they were not used because they disturbed leaflet movement in the closely related plant, Albizziajulibrissin (unpublished data; also, Satter et al. [8]).Immediately after the final wash in one of these solutions, protoplasts were mounted on sample supports made from the bottoms of aluminum weighing boats (Fisher Scientific). Aluminum was used because it has a low atomic number, high heat conductivity, and is easier to handle than thin pieces of plastic, or plastic films. The inside surface of the bottom of a weighing boat was coated with 0.5 ml 0.2% formvar (w/v) in dichloroethane and drained. The bottom of the weighing boat (0.09-0.1 mm thick) was then cut into squares about 2 mm on a side. A 2 Al droplet of 0.2% (w/v) polylysine in 80% (v/v) ethanol was layered over the formvar on each square and allowed to dry. One droplet of protoplast suspension (3-4 ,ul) was placed on each square. Protoplasts were allowed to settle, and the droplet was drained offwith Whatman No. 3 filter paper, leaving protoplasts adhering to the polylysine. Each square of foil with its adhering protoplasts was immediately frozen in liquid freon 22 (cooled to -145 to -150°C with liquid nitrogen) and lyophilized. Ten to 15 replicate samples were prepared in this way from each population of extensor and flexor protoplasts. Ten to 15 min elapsed between the time the cold, final wash was started and the time the last sample was frozen. The lyophilized samples were stored over P205 under light vacuum.As many as 10 aluminum squares, each bearing a population of lyop...

Section: Discussionsupporting

confidence: 66%

Extensor and Flexor Protoplasts from Samanea Pulvini

Gorton¹,

Satter²

1984

“…2 in Ref. 22). The terminal, secondary pulvini, which subtend the most distal pair of pinnae, have been studied most extensively.…”

Section: Abstracimentioning

confidence: 91%

Water Relations in Pulvini from Samanea saman

Gorton

1987

Biological Sciences Group U-42, University ofConnecticut, Storrs, Connecticut 06268 ABSTRACI The movement of Samwaea leaflets depends upon changes in the curvature of the pulvinus at the base of each leaflet. Pulvinar bending and straightening, in turn, are driven by the movement of water between opposing (extensor and flexor) sides of the pulvinus. Although water movement depends on water potential (1) and thus on osmotic potential (X) and hydrostatic pressure (P), none of these parameters have been measured in Samauea. In this investigation, il and X were measured and P was calculated for extensor Leaves and leaflets of the nyctinastic, leguminous tree Samanea saman (Jacq.) Merrill3 are usually spread horizontally during the day and folded tightly into a vertical orientation at night (2,20). These movements are brought about by the bending and straightening ofcylindrical motor organs, or pulvini, that subtend the leaf, the pinnae and the pinnules (see Fig. 2 (12,13,19). However, the magnitude and direction of the H+ fluxes varies with the 74 of the medium (16). These results are suggestive of a turgor-sensing mechanism, but they are difficult to interpret because we know very little about the I on the extensor and flexor sides of the pulvinus. If these in vitro experiments are to have maximum relevance to events occurring in whole pulvini, we must be able to design an appropriate bathing medium, with 7r balanced against the tissue , and we must understand the effects of excision.Pulvinar bending depends upon water movement, and thus on the I, wr, and P on the two sides of the pulvinus. regime at 27°C, as described in Gorton and Satter (10). Terminal secondary pulvini were used for all experiments. A protractor was used to measure leaflet angles (the angle between the rachis and the rachilla).Osmolality Measurements. Fluid from extensor and flexor tissues was obtained by two methods.1. Strips ofextensor and flexor tissue were excised as described in Iglesias and Satter (12). The tissue strips were squashed between a glass slide and a stainless steel spatula, and expressed fluid was collected with a glass microcapillary, all within a 4Abbreviations: ir = osmotic potential; PEG = polyethylene glycol (mol wt 3350); P = hydrostatic pressure; 1 = water potential (+ = r + P). 945www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

“…These changes in cell volume depend in turn on osmotic water uptake or water loss that follows ion influx or efflux from cells in the extensor and flexor regions of the pulvinus (6,24). Concentrations of K+, the most abundant ion in the pulvinus, increase approximately fourfold in extensor cells upon leaflet opening (14). Opening and closing of the leaflets and the ion fluxes that cause these movements are rhythmic, being under the control of an endogenous circadian clock (16,24).…”

Section: K+ Transport In Turgor Regulationmentioning

confidence: 99%

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

Kim

Coté²,

Crain³

1992

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.