Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 leve ls close them. This CO2 response is maintained in the dark. To elucidate me chanisms underlying the dark CO2 response we introduced pH- and potential-s ensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiratio n were simultaneously monitored. Upon an increase in CO2 concentration tran sient changes in apoplastic pH (occasionally brief acidification, but alway s followed by alkalinization) and in membrane potential (brief hyperpolariz ation followed by depolarization) accompanied stomatal closure. Alkalinizat ion and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening i n response to CO2-free air the apoplastic pH increased while the membrane p otential initially depolarized before it transiently hyperpolarized. To exa mine whether changes in apoplastic malate concentrations represent a closin g signal for stomata, malate was fed into the transpiration stream. Althoug h malate caused apoplastic alkalinization and membrane depolarization remin iscent of the effects observed with CO2 and abscisic acid, this dicarboxyla te closed the stomata only partially and less effectively than CO2. Apoplas tic alkalinization was also observed and stomata closed partially when KCI was fed to the leaves. Respiration increased on feeding of malate or KCl, o r while abscisic acid closed the stomate. From these results we conclude th at CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H-ATPases during changes in stomatal aperture. Responses to po tassium malate and KCI are not restricted to guard cells and neighbouring c ells.