Ca(2+)-independent and Ca(2+)/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 Ca(2+) and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 μM) free Ca(2+) 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 Ca(2+), 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 Ca(2+) 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 Ca(2+)-independent and whose regulation is currently not known and another that is stimulated by Ca(2+) and modulated by GTP-binding proteins. We suggest that Ca(2+)-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca(2+)-stimulated exocytosis may play a role in gibberellic acid-stimulated α-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.

胞吐作用（Exocytosis）能够介导分泌产物的释放，并将新生膜物质递送至质膜。截至目前，学界对植物细胞中胞吐作用的潜在分子机制及其调控过程仍知之甚少。本研究采用全细胞膜片钳技术（whole-cell patch-clamp technique），通过监测膜电容（membrane capacitance）变化，对大麦糊粉层原生质体（barley aleurone protoplasts）中的胞吐作用展开研究。为探究钙离子（Ca²+）与GTP结合蛋白（GTP-binding proteins）是否参与胞吐作用，我们分别使用极低浓度（<2 nM）与高浓度（1 μM）的游离钙离子，并搭配不可水解鸟嘌呤核苷酸：鸟苷5′-[γ-硫代]三磷酸（GTP[γS]）或鸟苷5′-[β-硫代]二磷酸（GDP[βS]），对原生质体进行胞内灌流透析。当胞质游离钙离子浓度低于2 nM时，膜电容在20分钟内出现显著升高，且该升高过程不受GTP[γS]或GDP[βS]的影响。与之相反，当使用1 μM游离钙离子进行胞内灌流透析时，膜电容出现大幅升高；该升高过程可被GTP[γS]轻度抑制，且被GDP[βS]显著阻断。本研究结论表明，大麦糊粉层原生质体中存在两条胞吐通路：一条为钙离子非依赖型，其调控机制目前尚未明确；另一条则受钙离子激活，并由GTP结合蛋白进行调控。我们推测，钙离子非依赖型胞吐作用可能参与发育中原生质体的细胞扩张过程；而钙离子激活型胞吐作用则可能在大麦糊粉层中参与赤霉素（gibberellic acid）诱导的α-淀粉酶（α-amylase）分泌，更广泛地说，其可能参与细胞损伤后的膜修复过程。