Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1

Abstract

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.

Figure 1

uvr8-1 is hypersensitive to UV-B in comparison with wild-type Ler. Wild-type Ler and uvr8-1 plants were grown in the absence of UV-B for 10 d. They were then treated with 72 h of 0.2 kJ UV-B_BE m⁻² h⁻¹ (+) and were allowed a 72-h recovery period in white light without UV-B. Identically aged control plants were grown in the absence of UV-B throughout the experiment (−). Plants were photographed immediately after the UV-B treatment or following a 72-h recovery period. A, Plants photographed immediately after a 72-h UV-B treatment. B, Plants photographed after a 72-h recovery period following a 72-h UV-B treatment.

Figure 2

uvr8-1 reduces the UV-B-mediated induction of flavonoid accumulation. Plants were grown in the absence of UV-B for 14 d and were treated with 0.4 kJ UV-B_BE m⁻² h⁻¹ for 3 d. Control plants were grown under white light in the absence of UV-B. Leaf tissue was harvested and methanol extracts were fractionated by reverse-phase HPLC. Numbers indicate the sinapate esters (peaks 5 and 6) and flavonoids (peaks 7–10), as previously identified by Li et al. (1993). A, Chromatogram of extract from Ler untreated control. B, Chromatogram of extract from uvr8-1 untreated control. C, Chromatogram of extract from Ler treated with UV-B for 3 d. D, Chromatogram of extract from uvr8-1 treated with UV-B for 3 d.

Figure 3

Induction of CHS, PR1, and PR5 proteins by UV-B. Plants were grown in the absence of UV-B for 14 d and were then treated with 0.4 kJ UV-B_BE m⁻² h⁻¹ for 1, 2, or 3 d. Control plants were grown to the same age in the absence of UV-B. A, Immunoblot with CHS antisera. Control plants showed no detectable CHS protein. B, Immunoblot with PR1- and PR5-specific antisera.

Figure 4

Analysis of CHS mRNA induction following UV-B treatment. Reverse transcriptase (RT)-PCR analysis of CHS mRNA induction by UV-B. Fourteen-day-old plants were grown in the absence of UV-B and were treated with 0.4 kJ UV-B_BE m⁻² h⁻¹ for 24 h or were left under −UV-B conditions for an additional 24 h. Tissue was then harvested for RNA extraction. Ethidium bromide-stained products are shown from quantitative RT-PCR. For each sample, 1 and 2 μg of total RNA were used for cDNA synthesis prior to PCR amplification. The results are representative of three independent experiments.

Figure 5

Chromosome walk to uvr8-1 locus. A, Markers used for localizing uvr8-1 to chromosome 5. Numbers at top represent genetic distance to uvr8-1 in centromeres. B, The four P1 clones (MBK5, MGI19, MVD12, and MHJ24) covering the uvr8-1 locus, with their sizes indicated in parentheses. The physical positions of markers MBK5-1 (A), MBK5C3 (B), MGI19-1 (C), MGI19C7 (D), MGI19C9 (E), MGI19C8 (F), MGI19C6 (G), MHJ24C1 (H), and MHJ24-2 (I) are indicated. C, Map of approximate locations of recombination breakpoints used for fine mapping. D, Smallest region genetically identified to contain uvr8-1 mutation. The five ORFs are putative amino acid transporter (1), RCC1 homolog (2), Ser/Thr protein phosphatase (3), hypothetical protein (4), and histidinol dehydrogenase (5). E, DNA sequence of the uvr8-1 15-bp deletion.

Figure 6

Deletion in uvr8-1 removes a Gly conserved among RCC1 homologs. An alignment of the 15 amino acids surrounding the uvr8-1 deletion. The sequences are UVR8, uvr8-1, RCC1 from human (Homo sapiens; U50078), and homologs from hamster (P23800), Xenopus (D00646), fruit fly (Drosophila melanogaster; S15028), and yeast (Saccharomyces cerevisiae; P21827). RLD2 is a human protein that contains a domain similar to the entire RCC1 protein (Rosa et al., 1996). Asterisks mark the uvr8-1 deletion. The numbers designate the distance from the carboxyl terminus. Gly described in the text are in bold.

Figure 7

Generation of the UVR8 complementation construct. Boxes represent ORFs with gene labels contained within each box. Numbers at the top represent location of restriction enzyme digestion sites in kilobases. A, HindIII fragment containing the putative amino acid transporter and UVR8. B, Final HindIII cassette containing UVR8 obtained after removing the NcII fragment containing the amino acid transporter. C, Restriction map of pGPTV-BAR binary vector. L, Left T-DNA border; R, right T-DNA border.