Ca²⁺-independent and Ca²⁺/GTP-binding protein-controlled exocytosis in a plant cell

Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca²⁺ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 μM) free Ca²⁺ and nonhydrolyzable guanine nucleotides guanosine 5′-γ-thio]triphosphate (GTP[γS]) or guanosine 5′-[β-thio]-diphosphate (GDP[βS]). With less than 2 nM cytoplasmic free Ca²⁺, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP [γS] or GDP[βS]. In contrast, dialyzing protoplasts with 1 μM free Ca²⁺ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[γS] and strongly inhibited by GDP[βS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca²⁺-independent 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 α-amylase secretion in barley aleurone and, more generally, may be involved in membrane reseating in response to cell damage.