Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.
BACKGROUND
Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.


RESULTS
Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.


CONCLUSIONS
One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.
Elgass
Kirstin
K
Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.
Caesar
Katharina
K
Schleifenbaum
Frank
F
Stierhof
York-Dieter
YD
Meixner
Alfred J
AJ
Harter
Klaus
K
D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000378:metabolism; D060406:Brassinosteroids; D002468:Cell Physiological Phenomena / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002777:Cholestanols / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018546:Hypocotyl / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013260:Steroids, Heterocyclic / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D002777:Cholestanols; D011993:Recombinant Fusion Proteins; D013260:Steroids, Heterocyclic; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C108270:BRI1 protein, Arabidopsis; C023623:brassinolide