Department of Agrotechnology and Food Sciences, Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
Plant Physiol. 2011 Aug;156(4):1691-700. doi: 10.1104/pp.111.179309. Epub 2011 May 26.
In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors μm⁻² in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors μm⁻². Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor density.
在植物中，绿色荧光蛋白（GFP）通常被用于确定融合蛋白的亚细胞定位。在这里，我们展示了共聚焦成像可用于估算活拟南芥（Arabidopsis thaliana）根细胞中 GFP 标记蛋白分子的数量。该技术涉及使用可溶性 GFP 进行校准，以提供在显微镜共聚焦体积内可用的蛋白浓度范围。作为原理验证，我们对受自身启动子控制的 GFP 融合的 Brassinosteroid Insensitive1（BRI1）受体进行了定量。每个根表皮细胞中的 BRI1-GFP 分子数从分生组织中的 22000 个到伸长区中的 130000 个到成熟区中的 80000 个不等，表明 BRI1 受体含量存在高达 6 倍的差异。相比之下，当考虑到细胞大小的差异时，在整个分生组织和伸长区的所有细胞中，BRI1-GFP 受体在质膜中的密度保持不变，为 12 个受体/μm²。只有静止中心和柱状细胞偏离这种模式，其密度为 5 到 6 个受体/μm²。值得注意的是，根细胞对油菜素甾体的敏感性似乎与均匀的分生组织受体密度一致。
