Pandey Sona, Wang Xi-Qing, Coursol Sylvie A, Assmann Sarah M
Biology Department, The Pennsylvania State University, 208 Mueller Laboratory, University Park, PA, USA, 16803.
New Phytol. 2002 Mar;153(3):517-526. doi: 10.1046/j.0028-646X.2001.00329.x. Epub 2002 Mar 5.
• Guard cells play an important role in the physiology and development of plants. The genetic resources available for Arabidopsis thaliana make it the most favorable plant species for the study of guard cell processes, but it is not easy to isolate highly purified preparations of large numbers of guard cells from this species. Here, we describe methods for isolation of both guard cell and mesophyll cell protoplasts from A. thaliana and their use in the study of unique biochemical and cellular properties of these cell types. • Protocols developed for large- and small-scale preparation of guard cell protoplasts and mesophyll cell protoplasts are described, followed by specific examples of their use in electrophysiological, biochemical and molecular approaches such as patch clamping, enzyme assays, and reverse-transcription polymerase chain reaction. • The protocols described yield millions of highly purified, viable guard cell protoplasts and mesophyll cell protoplasts from A. thaliana. These protoplasts have been used successfully in the study of ion channel properties, assay of ABA activation in phospholipase D activity and comparisons of gene and protein expression levels. • These techniques make it possible to elucidate electrophysiological, biochemical and molecular genetic pathways of guard cell function.
• 保卫细胞在植物的生理和发育过程中发挥着重要作用。拟南芥所具备的遗传资源使其成为研究保卫细胞过程最适宜的植物物种，但从该物种中分离出大量高度纯化的保卫细胞制剂并非易事。在此，我们描述了从拟南芥中分离保卫细胞和叶肉细胞原生质体的方法，以及这些方法在研究这些细胞类型独特的生化和细胞特性方面的应用。
• 文中描述了用于大规模和小规模制备保卫细胞原生质体和叶肉细胞原生质体的实验方案，随后列举了它们在电生理、生化和分子方法（如膜片钳、酶测定以及逆转录聚合酶链反应）中的具体应用实例。
• 所述实验方案可从拟南芥中获得数百万个高度纯化的、有活力的保卫细胞原生质体和叶肉细胞原生质体。这些原生质体已成功应用于离子通道特性研究、磷脂酶D活性中ABA激活的测定以及基因和蛋白质表达水平的比较。
• 这些技术使得阐明保卫细胞功能的电生理、生化和分子遗传途径成为可能。
