Abstract
To understand molecular processes in living plant cells, quantitative spectro-microscopical methods are required. We combined fluorescence lifetime and wavelength-specific spectroscopy with high resolution confocal microscopy to study the subcellular properties and function of a GFP-tagged version of the plasma membrane-bound steroid hormone receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis and tobacco plants. Plant steroids (brassinosteroids) regulate many aspects of plant (elongation) growth and development. Shortly after adding brassinolide (BL), a prototypical brassinosteroid, we observed a BRI1-dependent cell wall expansion at nm-scale proceeding cell elongation growth. Even a few minutes earlier, the fluorescence lifetime (FLT) of BRI1-GFP decreased, indicating an alteration in the receptor´s physico-chemical nano-environment. The parameter modulating the FLT of BRI1-GFP was found to be a hormone-induced hyperpolarisation of the plasma membrane, involving the activation of the plasma membrane H+ pump (P-ATPase). This activation required an interaction of BRI1 with the pump as demonstrated by a novel Fluorescence Resonance Energy Transfer method (FRET-FIDSAM). Our results can be combined in a model, which suggests that there is a brassinsteroid-regulated signal response pathway within the plasma membrane that links BRI1 with the proton pump for the regulation of cell wall expansion and cell elongation growth in plant cells. Furthermore, our approach shows how the experimental combination of high resolution confocal microscopy and spectroscopy reveals a new kind of data necessary for the quantitative description of subcellular processes in living plant cells.