Center for Plant Molecular Biology, Department of Plant Physiology, University of Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany.
Plant J. 2011 May;66(3):528-40. doi: 10.1111/j.1365-313X.2011.04510.x. Epub 2011 Mar 1.
To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.
为了理解活体植物细胞中的分子过程，需要定量光谱显微镜技术。通过将荧光寿命光谱学与共焦显微镜相结合，我们研究了拟南芥幼苗活细胞中质膜结合油菜素内酯受体 BRI1（BRI1-GFP）的 GFP 标记变体的亚细胞特性和功能。在添加油菜素内酯后不久，我们观察到 BRI1 依赖性的细胞壁扩张，先于细胞伸长。同时，BRI1-GFP 的荧光寿命降低，表明受体的物理化学环境发生了变化。调节 BRI1-GFP 荧光寿命的参数被发现是油菜素内酯诱导的质膜超极化。此外，为了诱导超极化和细胞壁扩张，需要激活质膜 P-ATP 酶。这种激活需要 BRI1 激酶活性，并通过油菜素内酯调节的 BRI1 与 P-ATP 酶的相互作用介导。我们的结果被用于开发一个模型，该模型表明，在质膜内存在一个快速的油菜素内酯调节的信号响应途径，该途径将 BRI1 与 P-ATP 酶联系起来，以调节细胞壁扩张。
