Regulation of Calcium Transport in Barley Aleurone Cells

提出的研究目标是实现对大麦糊粉细胞钙稳态的理解。该实验室先前对含钙金属酶α -淀粉酶合成的研究表明，当钙在细胞质中不断流动时，细胞质中的钙浓度保持在一个狭窄的范围内。提出了实验来确定钙的来源，保证细胞质钙稳态。膜片钳技术将用于鉴定糊粉细胞质膜和质质体中调节离子进入细胞质的通道。实验方法依赖于从糊粉层分离功能原生质体的能力，并利用这些原生质体作为完整液泡的来源。本实验室已测定了糊粉质原生质体质膜和从原生质体分离出来的液泡中的通道活性；因此，这种方法应该会成功。在种子萌发过程中，储存在贮藏器官（如糊粉层）液泡中的代谢物被动员起来供生长中的植物胚胎使用。尽管这些储存的储备是至关重要的，但人们对这些物质是如何调动的知之甚少。据认为，赤霉素与此有关，但其作用机制尚不清楚。本实验室已经证明，钙的动员对赤霉素酸诱导的α -淀粉酶的合成至关重要，α -淀粉酶是动员糊粉细胞中储存产物的重要酶。该项目旨在了解钙是如何在大麦糊粉细胞中调节的。这一结果阐明了种子萌发过程中代谢物动员调控的一个重要步骤。

The goals of the proposed research are to achieve an understanding of calcium homeostasis in the barley aleurone cell. Prior work from this laboratory on the synthesis of the calcium- containing metalloenzyme, alpha-amylase, has established that the cytoplasmic calcium concentration is maintained within narrow limits while a constant flux of calcum occurs through the cytoplasm. Experiments are proposed to determine the source of the calcium that assures cytoplasmic calcium homeostasis. Patch clamp techniques will be used to identify the channels in the plasm membrane and tonoplast of the aleurone cell that regulate the flux of ions into the cytoplasm. The experimental approach relies on the ability to isolate functional protoplasts from the aleurone layer and to use these protoplasts as a source of intact vacuoles. The activties of channels in the plasma membrane of aleurone protoplasts and in vacuoles isolated from protoplasts have already been measured in this laboratory; thus, this approach should succeed. During seed germination metabolites stored in the vacuoles of storage organs such as aleurone layers are mobilized for use by the growing plant embryo. Although these stored reserves are crucial, little is known about how these materials are mobilized. It is thought that giberellic acid is involved, but the mechanism by which it acts is not known. This laboratory has shown that mobilization of calcium is of central importance to the giberellic acid-induced synthesis of alpha-amylase, an enzyme important in mobilizing storage products in the aleurone cell. This project is directed toward understanding how calcium is regulated in barley aleurone cells. The results should elucidate an important step in the regulation of metabolite mobilization during seed germination.