Molecular Cloning and Functional Analysis of UV RESISTANCE LOCUS 8 (PeUVR8) from Populus euphratica

Abstract

Ultraviolet-B (UV-B; 280-315 nm) light, which is an integral part of the solar radiation reaching the surface of the Earth, induces a broad range of physiological responses in plants. The UV RESISTANCE LOCUS 8 (UVR8) protein is the first and only light photoreceptor characterized to date that is specific for UV-B light and it regulates various aspects of plant growth and development in response to UV-B light. Despite its involvement in the control of important plant traits, most studies on UV-B photoreceptors have focused on Arabidopsis and no data on UVR8 function are available for forest trees. In this study, we isolated a homologue of the UV receptor UVR8 of Arabidopsis, PeUVR8, from Populus euphratica (Euphrates poplar) and analyzed its structure and function in detail. The deduced PeUVR8 amino acid sequence contained nine well-conserved regulator of chromosome condensation 1 (RCC1) repeats and the region 27 amino acids from the C terminus (C27) that interact with COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC1). Secondary and tertiary structure analysis showed that PeUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis thaliana. Using heterologous expression in Arabidopsis, we showed that PeUVR8 overexpression rescued the uvr8 mutant phenotype. In addition, PeUVR8 overexpression in wild-type background seedlings grown under UV-B light inhibited hypocotyl elongation and enhanced anthocyanin accumulation. Furthermore, we examined the interaction between PeUVR8 and AtCOP1 using a bimolecular fluorescence complementation (BiFC) assay. Our data provide evidence that PeUVR8 plays important roles in the control of photomorphogenesis in planta.

Fig 1. Hydrophilicity/hydrophobicity analysis and PeUVR8 transmembrane domain prediction.

(A) Hydrophilicity/hydrophobicity analysis of PeUVR8 performed using the Kyte and Doolittle method. A score of 1.8 is indicated by the red line. (B) PeUVR8 transmembrane domain prediction using the DNAMAN version 5.2.2 software with default options. Predicted transmembrane regions are indicated by boxes above the profile.

Fig 2. PeUVR8 protein sequence analysis.

(A) Structural domains of the PeUVR8 and AtUVR8 proteins. Analysis of protein sequences in the National Center for Biotechnology Information (NCBI) database was performed using the CD-search software. (B) Amino acid sequence alignment of UVR8 proteins from Euphrates poplar, Arabidopsis, Populus trichocarpa, apple, castor, and rice. The alignment was constructed using the DNAMAN version 5.2.2 software. Identical residues are indicated by black boxes. Red dots above the sequences indicate the 14 conserved tryptophan residues and the red frames indicate the triad tryptophans (positions 233, 285, 337 in the Arabidopsis sequence) that form a pyramid arrangement with W94 on the adjacent monomer. Black dots above the sequences indicate the two arginine residues (positions 286, 338 in the Arabidopsis sequence) that are key to maintaining the AtUVR8 dimer. Red lines under the sequences indicate the conserved “GWRHT” motifs and the green box indicates the conserved C27 region. Purple lines indicate the seven blades compared with AtUVR8. Grey lines under the AtUVR8 sequence indicate the missing residues in the AtUVR8 crystal structure.

Fig 3. Structural analysis of the PeUVR8 protein.

(A) The secondary structure of PeUVR8 solved using the self-optimized prediction method (SOPM). Red dots above the sequence indicate the 14 conserved tryptophans. Red lines and black dots indicate the crucial two tryptophans (W233, W285) and two arginines (R286, R338) corresponding to AtUVR8, respectively. Purple lines indicate the seven blades of PeUVR8 compared with AtUVR8. (B) Comparison of the predicted three-dimensional structures of PeUVR8 and AtUVR8 using Cn3D software. Views from the top (above image) and from the side (image below). Red and green balls in the images indicate the conserved tryptophans with the red balls indicating the four crucial tryptophans (W94 and the clustered triad tryptophans W233, W285, W337) used to form pyramids between the UVR8 dimer. White balls indicate the two arginines (R286, R338) that are crucial for maintaining the dimer structure of UVR8. The seven blades of UVR8 are represented by different colors (under the two images). (C) 3D structure of the UVR8 dimer (viewed from the side). Red and yellow balls indicate the two pyramids between the two monomers.

Fig 4. Local disorder tendency prediction and fold disordering character analysis of PeUVR8 protein.

(A) Local disorder tendency of PeUVR8 based on an estimated-amino-acid-pairwise-energy-content analysis using the IUPred software. (B) The fold disordering character of PeUVR8 predicted using the FoldIndex software.

Fig 5. Phylogram of plant UVR8 proteins.

UVR8 amino acid sequences from 22 diverse plant species were obtained from the NCBI database. The alignment was constructed using ClustalX and the phylogenetic tree was constructed using the neighbor-joining method in the MEGA version 4.1 software. Each node corresponds to a number indicating the bootstrap value for 1000 replicates. The scale bar represents 0.1 substitutions per sequence position. PeUVR8 is denoted by a red dot. Pv, Phaseolus vulgaris; Gm, Glycine max; Ca, Cicer arietinum; Cm, Cucumis melo; Bp, Betula platyphylla; Pt, Populus trichocarpa; Pe, Populus euphratica; Md, Malus domestica; Pb, Pyrus bretschneideri; Pm, Prunus mume; Pp, Prunus persica; Cc, Citrus clementina; Cs, Citrus sinensis; Br, Brassica rapa; At, Arabidopsis thaliana; Cr, Capsella rubella; Es, Eutrema salsugineum; Th, Thellungiella halophila; Ma, Musa acuminate; Ob, Oryza brachyantha; Os, Oryza sativa; Si, Setaria italic; Zm, Zea mays.

Fig 6. Analysis of tissue-specific PeUVR8 expression.

Analysis of PeUVR8 expression in various tissues (roots, stems, leaves, shoots and buds) using (A) real-time quantitative and (B) semiquantitative RT-PCR. The PeACT gene was performed as an internal control. Error bars indicate s.e. over three biological replicates (each replicate, three technical replicates).

Fig 7. Phenotypes of wild-type (WT), uvr8-1 mutant, PeUVR8-transgenic uvr8-1 mutant, and PeUVR8-transgenic WT plants.

(A) PeUVR8 transcript levels in 4-day-old seedlings of WT, uvr8-1 mutant, and transgenic lines. Three uvr8-1 mutant lines transformed with PeUVR8 are designated m-1, m-2, and m-3. Three WT lines transformed with PeUVR8 are designated W-1, W-2, and W-3. (B) Phenotypes of the WT, uvr8-1 mutant, and two transgenic lines (m-2 and W-1) grown under white light with or without narrowband UV-B light. Scale bar represents 5 mm. (C) and (D), Quantitative RT-PCR analysis of HY5 and CHS gene expression levels in the WT, uvr8-1 mutant, and the two transgenic lines with different durations of UV-B irradiation. Error bars indicates s.e. over three biological replicates (each replicate, 10–15 pooled seedlings). (E) Hypocotyl lengths of 4-day-old seedlings of WT, uvr8-1 mutant, and two transgenic lines grown under white light with or without narrowband UV-B light. Error bars indicates s.d. (n > 30). (F) Anthocyanin content of 4-day-old seedlings of WT, uvr8-1 mutant, and two transgenic lines grown under white light with narrowband UV-B light. Error bars indicates s.d. (n > 30).

Fig 8. PeUVR8 interaction with AtCOP1 in bimolecular fluorescence complementation (BiFC) assays.

The right images are the overlay of YFP fluorescent (left) and bright-field (middle) images of onion epidermal cells cotransformed with indicated combinations. Y^N and Y^C represent the N- and C-terminal regions of YFP respectively.