Direct Determination of Potassium Ion Accumulation in Guard Cells

Understanding the mechanism of stomatal opening in leaves is important because stomata are the avenues for CO2 and H,O diffusion. Stomata in most species open in the light when the guard cells that encompass the pores take up water and increase in turgor relative to adjacent epidermal cells (1). Thus stomatal opening is likely an osmotic plhenomenon that depends upon the accumulation of solute in the guard cells, and during opening this accumulation has been reported to range from 0.' X to 0.5 M in various experiments (2). For over 100 years botanists have believed that the primary solute accumulating in the liglht was soluble carbohydrate produced during photosynthesis, because guard cells possess chloroplasts and otlier epidermal cells do not. However careful quantitative work [reviewed in (3)] failed to provide strong support for this view. Recent independent evidence from 2 laboratories suggests that the accumulation of potassiunm ionls in guard cells provides the essential solute for stomatal opening. Fujino (4) used cytochemical staining methods and found that open stomata in several species contained more potassium in their guard cells than closed stomata. Opening of stomata in epidermal strips of Commtelina comnlimunis in the liglht was stimulated by added potassium salts, anid quialitative tests showed that the potassium concentration increased in the guard cells. Fischer (5-7) slhowed that potassium and rubidium salts stimulated opening in epidermal strips of Vicia faba. Calculations made from the uptake of 80Rb' by such tissues indicated that if all of the accumulation were in the guard cells there would be sufficient solute (witlh an accompanying anion) to account for stomatal opening. Thus, although precise quantitative data were lacking, it seemed plausible that stomatal opening was mediated by a potassium ion punmp that accutmulated these ions in the guard cells (8). We have carried out a quantitative test of this hypothesis on intact leaf tissue by taking advantage of the electron microprobe teclhnique, wlhich permits an accurate determination of the concentration of an element in a 1 to 2 u diameter region (9). Essentially, a beam of electrons of desired diameter is focused on the specimen surface. The clhemical elements irradiated by the beam emit tlleir characteristic X-ray spectra. Tlle emitted X-rays are then analyzed according to wavelength and intensitv, permitting both qualitative and quantitative chemical analysis of micron-sized volumes. Tobacco leaves (Nicoliana tabacum, variety Havana Seed) were excised from greenlhouse grown plants and placed in darkness for several hr so that the stomata closed. Leaf disks 3.2 cm in diameter were cut with a sharp punch and floated upside down on water in the air at 30° in 1000 ft-c illunmination. Following illuminiation the disks were left in the dark for various periods. After these treatments, portions of the lower epidermis were quickly stripped off. They were placed on a glass slide and rapidly