CA2-INDEPENDENT AND CA2+()GTP-BINDING PROTEIN-CONTROLLED EXOCYTOSIS IN A PLANT-CELL/

Exocytosis allows the release of secretory products and the delivery o f new membrane material to the plasma membrane. So far, little is know n about the underlying molecular mechanism and its control in plant ce lls. We have used the whole-cell patch-clamp technique to monitor chan ges in membrane capacitance to study exocytosis in barley aleurone pro toplasts. To investigate the involvement of Ca2+ and GTP-binding prote ins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 mu M) free Ca2+ and nonhydrolyzable guanine nucleotides guano sine 5'-gamma-thio]triphosphate (GTP[gamma S]) or guanosine 5'-[beta-t hio]diphosphate (GDP[beta S]). With less than 2 nM cytoplasmic free Ca 2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gamma S] or GDP[beta S]. In contrast, dialyzing protoplasts with 1 mu M free Ca2+ resulted in a large incre ase in membrane capacitance that was slightly reduced by GTP[gamma S] and strongly inhibited by GDP[beta S]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-indep endent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in respons e to cell damage.