Guard Cells Extrude Protons Prior to Stomatal Opening—A Study using Fluorescence Microscopy and pH Micro-electrodes

A method for the demonstration of pH changes in the apoplast is described. The fluorescent pH indicator pnmulin was used to follow pH changes in the epidermis of leaves of Commelina communis during stomatal movements. Previously darkened leaves exposed to light showed quenching of fluorescence in the apoplast surrounding theǵuard cells up to 20 min before the stomata opened. This indicated that proton efflux by the guard cells preceded stomatal opening. This result was substantiated by apoplastic pH measurements using pH micro-electrodes. Acidification of the apoplast spread outwards from the guard cells to the surrounding subsidiary and epidermal cells. This phenomenon persisted for some time after subsequent stomatal closure, supporting the hypothesis that closure is brought about by a process other than the cessation of proton pumping.