Ruohe Yin, Mariya Y. Skvortsova, Sylvain Loubéry, and Roman Ulm

Significance

Plant tissues are resistant to the potentially damaging UV-B radiation intrinsic to sunlight. UV-B photoreception by a UV RESISTANCE LOCUS 8 (UVR8) photoreceptor regulates gene expression in plants associated with UV-B acclimation and stress tolerance and with morphological changes. Mechanistically, UV-B photon reception by specific tryptophan residues of UVR8 homodimers results in monomerization and enhanced nuclear accumulation of UVR8. Active UVR8 monomers interact with the key signaling protein CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). This UV-B–dependent interaction is a crucial step in signal propagation, but the link between this mechanism and UVR8 nuclear accumulation and gene expression remains ill defined. Our results emphasize the importance of nuclear-localized UVR8 and highlight a previously unknown activity of COP1 in mediating UVR8 nuclear accumulation in response to UV-B.

Abstract

The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana. UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B–induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B–induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B–activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8^W285A accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B–activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B–induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling.

See http://www.pnas.org/content/113/30/E4415.abstract.html?etoc

PNAS July 26 2016; vol.113; no.30: E4415–E4422

Fig. 1.

Nuclear accumulation of UVR8 requires COP1. (A-C) Immunoblot analyses: (A) UVR8, histone H3 (nuclear control), and UGPase (cytosolic control) in cytosolic and nuclear proteins of 7-d-old wild-type plants (Col) grown in white light without (0 h) or with UV-B for 4 h or 24 h. (B) UVR8, histone H3, and UGPase in nuclear (Left) and cytosolic proteins (Right) of uvr8-6, wild-type (Col), cop1-4, and cop1-20 plants grown in white light without (−UV-B) or with UV-B for 6 h (+UV-B). (C) Total UVR8 protein levels of uvr8-6, wild-type (Col), cop1-4, and cop1-20 plants grown in white light without (−UV-B) or with UV-B for 6 h (+UV-B). (D) UVR8, YFP-UVR8, and actin (loading control) proteins in 4-d-old uvr8-6, wild-type (Col), uvr8-6/Pro_35S:YFP-UVR8 (uvr8-6/YFP-UVR8 3), and cop1-4/Pro_35S:YFP-UVR8 (cop1-4/YFP-UVR8 6 and 11) lines. (E) YFP and DAPI fluorescence in cotyledon adaxial epidermis of 4-d-old uvr8-6/Pro_35S:YFP-UVR8 line 3 and cop1-4/Pro_35S:YFP-UVR8 line 6. (Scale bars: 10 µm.) (F) Yeast two-hybrid growth assays of COP1 interactions with wild-type UVR8, UVR8^ΔN23 truncation, and constitutively active variant UVR8^W285A on selective –His medium (SD/-Trp/-Leu/-His) in the presence or absence of UV-B. Growth on +His medium (SD/-Trp/-Leu) as a transformation control. AD, activation domain; BD, binding domain. (G) Coimmunoprecipitation of COP1 using anti-GFP antibodies in extracts from uvr8-7 (negative control), uvr8-7/Pro_35S:YFP-NLS-UVR8, and uvr8-7/Pro_35S:YFP-NLS-UVR8^ΔN23 lines. Seven-day-old seedlings were treated with broadband UV-B for 15 min (+UV-B) or not (−UV-B). IB, immunoblotting; IP, immunoprecipitation (H and I) Immunoblot analyses: (H) UVR8, histone H3, and UGPase nuclear (Upper) and cytosolic proteins (lower) of 7-d-old uvr8-7, wild-type (Ws), uvr8-7/Pro_35S:UVR8^W285A line 4 (uvr8-7/UVR8^W285A), and cop1-4 uvr8-7/Pro_35S:UVR8^W285A (cop1-4 uvr8-7/UVR8^W285A) treated with 9-h narrowband UV-B or not. (I) UVR8, histone H3, and UGPase in nuclear (Upper) and cytosolic proteins (lower) of 7-d-old wild-type (Col) and uvr8-6, and rup1 rup2 treated with 6-h narrowband UV-B or not.