Opening of Stomata in Epidermal Strips'

The effect of various ions on stomatal opening was studied in isolated epidermal strips of Vicia faba L. Stomata in strips floating on 10 mm KCI and in CO.,-free air opened in light, closed in subsequent darkness, then opened fully again when illutminated. A lightactivated highly specific effect of K' (and Rb+) on opening was found. When strips were floated on high concentrations (50 or 100 meq/liter) of Li+, Na+ or Cs', stomata opened but light had very little effect on the concentrations required for opening. With K+, the opening produced in the dark was the same as with the other alkali ions. Light, however, lowered more than 100-fold the concentration of K+ required for maximal opening. Thus only the effect of K+ (and Rb+) was greatly accentuated by light. NH4+ and Mg2+ did not produce opening. No specific anion is required in association with K+. Opening was the same when Cl-, IBr, and NO<were used as counter ions, but was less when SO.2was used, particularly at higher concentrations and in the dark. The results are discussed in relation to the recent proposal that the basis for stomatal opening is Kuptake in amounts sufficient to act as an osmotic agent. This work also demonstrates, for the first time, a physiological process specifically requiring K+. Assuming that ion uptake is an integral part of stomatal opening, guard cells would appear to hiave an ion uptake mechanism of a degree of specificity previously unknown in higher plants. The mechaiiism of stomiiatal imiovemeient has been studied for imianyI decades. There is. however. still Ino general agreeimient on the process by which solute concentrations in guard cells are changed to effect ,chan'ges in turgor and the consequent stomatal move:ment. The classical starch-suigar hypotlhesis of Sayre (19) has been criticized extensively (11. 16) but still hats sotmie support (14)! The response of stom.ata in isolated epidermal strips to various inorganic ionls lias been extensively investigated by earlier workers. However, such stomiata did niot respond normally to light. Thus the results obtained were considered by Heath (8) to be inot necessarily relevant to the behavior of stomata in the intact leaf. The appalrent niornmal responise to natural stimuli by,stomnata in strips has been deemons;trated onlv recenitly (5, 7). Fischer i(5), in pre'ious w-ork in this laboratory, showed that the gutard cells in strips fromii Vicia faba L. were living as judged 1b acctumiiulation of neutral red aand the presence of protoplasmic streaming. They also responded to liglht and CO.-free air when the strips were floated onl KCI soltutions in the same mnannier as did the stomlata in leaf discs. This sili'Supported in part by grant B-029-CAI, from the Office of Water Resources Research, United States Departmenit of the Interior, an(d by a grant from the WVater Resour-ces Ceniter, University of Califormia. 230 larity applied to changes in aperture, guard cell solute poten! tial, and guard cell starch conitent. From studies of strips also showing normal responses. Fujino (7) concluded that stomatal opening is the result of uptake of K+ into guard cells. A similar conclusion was reached from work done indepenidently in our laboratory. Particularly, the data of Fischer and Hsiao (6) suiggest that the absorption of K+ and the associated increase in anion miay account for most of the changes in solute concentration in guard cells associated with stomatal openiing. \We now report on the specific requiremenit of K+ (or Rb+) for stomatal openinig in V. faba. Materials and Methods The isolated epiderm-ial strip technique of Fischer (5i) was used with slight modifications. The plants (V. faba, var. Long Pod) were grown for 3 to 5 weeks in a growth chamber at a light intensity higher than that tused in the earlier work (5). The conditions were a 14-hr day of approximately 3000 ft-c, a constant temperature of 26', and a relative humidity of approximately 70 %. Epidermal strips (approximately 3 X 5 mm) Nvere taken from the lower surface of the first to fourth fully expanded leaves and immediately floated ctuticle up for 35 mmi on 0.1 nvt CaCl. , soiultion inl complete darkness. Tllis enstured that virtually all www.plantphysiol. org on August 20, 2017 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved. HUMBLE AND HSIAO-SPECIFIC REQUIREMENT OF K FOR STOMATAL OPENING stomata were closed before experimental treatments were begun. The strips were then transferred to test solutions consisting of various ions buffered at pH 6 bv 0.5 mM trishydroxymethyl aminomethane/maleate/Ca2+ (5). In all experiments CO,-free air was passed continuously over the solutions (5). When light was used the intensity was 2200 to 2400 ft-c. Alternatively complete darkness was maintained. The solution temperatures were 30 to 340 as monitored with thermocouples. After floating on test solutions under these conditions for 3 hr, the strips were transferred to immmersion oil, and the width of aperture of 20 stomata, selected at random on each strip. was measured microscopically (5). It has been shown (5) that the apertures of stomata in imnmersion oil do not change for at least 30 min. Five or 6 replicates were used in an experiment. Strips from a single leaf constituted 1 replicate with 1 strip per treatment. Each valtue presented is an av-erage of at least 100 individual stomatal apertures. In experiments where cations other than K+ were tested at a range of concentrations for their effects on stomata, at least 1 concentration of KCI (usuall0.1 mM) was always included as a treatment to ascertain that the stomata used showed the typical response to K-.