Dimer/monomer status and in vivo function of salt-bridge mutants of the plant UV-B photoreceptor UVR8

Abstract

UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet-B (UV-B) light that initiates photomorphogenic responses in plants. UV-B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt-bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt-bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt-bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV-B responses in vivo with a similar dose-response relationship to wild-type. The UV-B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV-B photoreception, initiating signal transduction and responses in plants.

Keywords: Arabidopsis thaliana; UV-B; UVR8; photomorphogenesis; photoreceptor.

Figure 1

Dimer/monomer status of purified UVR8 salt‐bridge mutant proteins. (a) PyMol image showing inter‐monomer salt‐bridges formed between R286 and D96 and D107. (b) Size exclusion chromatography on a Superdex 200 column of purified wild‐type UVR8 and the UVR8^R286A, UVR8^R286K and UVR8^D96N,D107N mutant proteins exposed (dashed line) or not (solid line) to 1.5 μmol m⁻² sec⁻¹ narrowband UV‐B for 1 h. Elution points of marker proteins (in kDa) are shown at the top.

Figure 2

Dimer/monomer status of UVR8 salt‐bridge mutant proteins expressed in plants. (a) Western blot of whole cell extracts from uvr8‐1 plants expressing either GFP‐UVR8 or GFP‐UVR8 salt‐bridge mutants exposed (+) or not (−) to 4 μmol m⁻² sec⁻¹ narrowband UV‐B for 30 min. SDS‐loading buffer was added and samples were run on a 7.5% SDS–PAGE gel without boiling. An immunoblot was probed with anti‐UVR8 antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. The GFP‐UVR8 dimer and monomer are indicated. (b) Size exclusion chromatography profiles of immunoprecipitated wild‐type GFP‐UVR8 (WT) and salt‐bridge mutant fusions expressed in Nicotiana benthamiana plants. Vector with GFP alone was used as a control. For wild‐type GFP‐UVR8, extracts were illuminated (or not) with 4 μmol m⁻² sec⁻¹ narrowband UV‐B for 30 min. All other samples were not exposed to UV‐B. Eluates of immunoprecipitation assays with anti‐GFP beads were loaded onto a Superdex 200 column, and fractions 15–30 were used for standard SDS–PAGE and immunoblotting with an anti‐GFP antibody. (c) Western blot of whole cell extracts from uvr8‐1 plants expressing either GFP‐UVR8 (WT) or GFP‐UVR8 salt‐bridge mutants not exposed to UV‐B incubated with the cross‐linking reagent dithiobis (succinimidylpropionate) (DSP) in the absence (upper panel) or presence (lower panel) of β‐mercaptoethanol (β‐ME). SDS‐loading buffer was added and samples were run on a 10% SDS–PAGE gel without boiling. An immunoblot was probed with anti‐UVR8 antibody. The UVR8 dimer and monomer are indicated.

Figure 3

Interaction of UVR8 mutants with COP1 in vivo. Co‐immunoprecipitation of GFP‐UVR8 and COP1 in whole cell extracts obtained from uvr8‐1 plants transformed with either GFP‐UVR8 or GFP‐UVR8 salt‐bridge mutants exposed (+) or not (−) to 3 μmol m⁻² sec⁻¹ narrowband UV‐B for 3 h. Co‐immunoprecipitation assays were performed under the same conditions. Input samples (15 μg, IN) and eluates (IP) were run on a SDS–PAGE gel, and an immunoblot was probed with anti‐COP1 and anti‐GFP antibodies.

Figure 4

Functional complementation of uvr8‐1 by UVR8 salt‐bridge mutant proteins. (a) Hypocotyl lengths (±SE, n = 10) for 4‐day‐old wild‐type Ler, uvr8‐1, GFP‐UVR8 and the indicated transgenic lines of UVR8 salt‐bridge mutant seedlings grown in 1.5 μmol m⁻² sec⁻¹ white light (−UV‐B) supplemented with 1.5 μmol m⁻² sec⁻¹ narrowband UV‐B (+UV‐B). (b) RT‐PCR assays of HY5,CHS and control ACTIN2 transcripts in Ler (WT), uvr8‐1, and independent transgenic lines expressing either UVR8^R286A, UVR8^R286K or UVR8^D96N,D107N grown under 20 μmol m⁻² sec⁻¹ white light (−) and exposed to 3 μmol m⁻² sec⁻¹ broadband UV‐B for 4 h (+). (c) Expression of CHS protein in GFP‐UVR8, uvr8‐1 and the indicated transgenic lines of UVR8 salt‐bridge mutant plants grown and illuminated as in (b) for 7 days. Protein extracts were run on standard SDS–PAGE, and an immunoblot was probed with anti‐CHS antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control.

Figure 5

UV‐B dose–response of HY5 transcript accumulation in UVR8^D96N,D107N mutant compared with GFP‐UVR8. (a) qRT‐PCR measurements of HY5 transcripts in GFP‐UVR8 and GFP‐UVR8^D96N,D107N plants exposed to a range of doses of narrowband UV‐B. HY5 transcripts were normalised to control ACTIN2 transcript levels and fold‐induction is relative to the pre‐illumination dark transcript level. The data are the means (±SD) of three independent experiments. (b) Abundance of GFP‐UVR8^D96N,D107N and GFP‐UVR8 proteins in plants used for the three replicate experiments in (a). Protein extracts were subjected to SDS–PAGE and an immunoblot was probed with anti‐GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. Quantification of relative band intensities indicates that the mean level of GFP‐UVR8^D96N,D107N is approximately 25% that of GFP‐UVR8.

Figure 6

Bimolecular fluorescence complementation (BiFC) analysis of UVR8^D96N,D107N dimer/monomer status. (a) UVR8^D96N,D107N (UVR8‐DD) is expressed as a translational fusion with either the N‐ or C‐terminal region of YFP. Monomer interaction will lead to reconstitution of YFP and hence fluorescence. (b) Co‐transfection of plasmids expressing UVR8^D96N,D107N/N‐YFP and UVR8^D96N,D107N/C‐YFP in Nicotiana benthamiana. Confocal image of YFP fluorescence superimposed on bright‐field image of N. benthamiana epidermal cells. Plants were exposed to either UV‐B or no UV‐B following transfection. Arrows indicate nuclei. (c) Confocal images of control transfections: UVR8^D96N,D107N/N‐YFP and empty vector/C‐YFP (N+/C−); empty vector/N‐YFP and UVR8^D96N,D107N/C‐YFP (N−/C+); empty vector/N‐YFP and empty vector/C‐YFP (N−/C−). Arrows indicate nuclei.