Issue 2, 2019

A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

Abstract

The photoreceptor UVR8 has a pivotal role in mediating plant responses to UV-B wavelengths. Dimeric UVR8 dissociates into monomers following UV-B photoreception, and there is evidence that this process is accompanied by conformational changes that may facilitate interaction of UVR8 with other proteins to initiate signaling. Hence monitoring UVR8 dimer/monomer status and conformation is key to understanding UVR8 action. Here we have used Fluorescence Resonance Energy Transfer (FRET) to study these processes in both wild-type and mutant UVR8 proteins in vivo. UVR8 was fused to GFP and mCherry at the C- and N-termini, respectively and both the FRET efficiency and loss of GFP fluorescence after photobleaching were measured. In addition, measurements were made for UVR8 fused to either GFP or mCherry to eliminate intra-molecular FRET signals. The results indicate that dissociation of UVR8 dimer to monomer principally accounts for the loss of FRET signal for wild-type UVR8 and there is little evidence of a contribution from conformational change in vivo. Examination of plants expressing UVR8W285F and UVR8D96N,D107N are consistent with these mutant proteins being constitutively dimeric and monomeric, respectively. The methods employed here will be valuable for monitoring UVR8 dimer/monomer status in vivo in relation to signaling, and will facilitate characterization of dimer/monomer status and conformation of further UVR8 mutants.

Graphical abstract: A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

Supplementary files

Article information

Article type
Paper
Submitted
29 Oct 2018
Accepted
03 Dec 2018
First published
04 Dec 2018
This article is Open Access
Creative Commons BY license

Photochem. Photobiol. Sci., 2019,18, 367-374

A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

X. Liao, B. Zhang, M. R. Blatt and G. I. Jenkins, Photochem. Photobiol. Sci., 2019, 18, 367 DOI: 10.1039/C8PP00489G

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

Read more about how to correctly acknowledge RSC content.

Spotlight

Advertisements