A UV-B-specific signaling component orchestrates plant UV protection

Bobby A. Brown, Catherine Cloix, Guang Huai Jiang, Eirini Kaiserli, Pawel Herzyk, Daniel J. Kliebenstein, and Gareth I. Jenkins
PNAS December 13, 2005 102 (50) 18225-18230; https://doi.org/10.1073/pnas.0507187102

  1. Edited by Alexander N. Glazer, University of California System, Oakland, CA (received for review August 22, 2005)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    UVR8 acts specifically in the UV-B regulation of CHS expression. Shown are RT-PCR measurements of CHS and control ACTIN2 transcripts (lower and upper bands, respectively, A-E). (A) Wild-type, uvr8-1, uvr8-2, and one of the F1 progeny of uvr8-1 and uvr8-2 grown for 3 weeks in low fluence rate (25 μmol m-2·s-1) white light (L) and illuminated with ambient (3 μmol m-2·s-1) UV-B for 4 h (UB). (B) Plants grown as in A illuminated with UV-A (UA) for 6 h. (C) Four-day-old dark-grown (D) seedlings illuminated with far-red (FR) light for 6 h. (D) Plants grown as in A transferred to 7°C for 24 h. (E) Seedlings grown in darkness for 4 days with (+S) or without (-S) 2% sucrose.

  • Fig. 2.
    Fig. 2.

    HY5 is regulated by UVR8 and required for UV-B protection. (A) HY5 and control ACTIN2 transcripts measured by RT-PCR in wild-type and uvr8-2 plants grown for 3 weeks in 25 μmol m-2·s-1 white light (L) and illuminated with 3 μmol m-2·s-1 UV-B (UB) for 4 h or UV-A (UA) for 6 h. (B) HY5 and ACTIN2 transcripts (lower and upper bands, respectively) in 4-day-old dark-grown (D) seedlings illuminated with far-red (FR) light for the times indicated. (C) UV-B sensitivity assay. Wild-type, hy5-1, and uvr8-1 plants grown in 120 μmol m-2·s-1 white light for 12 days and then exposed to above-ambient (5 μmol m-2·s-1) UV-B with supplementary 40 μmol m-2·s-1 white light for 24 h. Plants were photographed after return to white light for 5 days.

  • Fig. 3.
    Fig. 3.

    GFP-UVR8 is present in the nucleus and associates with chromatin. (A) uvr8-2 plants expressing a 35S promoter::GFP-UVR8 fusion grown in low fluence rate white light and illuminated with 3 μmol m-2·s-1 UV-B for 2 h. Confocal image shows GFP fluorescence of epidermal cells. (Scale bar, 20 μm.) (B) Single cell showing the nucleus (arrowed); left, bright-field image and right, with GFP fluorescence superimposed. (Scale bars, 20 μm.) (C) Localization of fluorescence in wild-type plants expressing a control 35S promoter::GFP fusion. (Scale bar, 20 μm.) (D) Binding of E. coli-expressed GST-UVR8 to a calf thymus histone-agarose column. Western blots with anti-GST antibody showing GST-UVR8 (73 kDa) or control GST (26 kDa). Lane 1, protein applied to the columns; lane 2, unbound material that flowed through; lanes 3, 4, and 5, protein bound after a 5-min incubation, eluted with 0.1, 0.3, or 1.0 M NaCl, respectively. (E) Chromatin immunoprecipitation assay of DNA associated with GFP-UVR8. PCR of HY5 promoter (-331 to +23) and ACTIN2 DNA from 35S::GFP-UVR8 (Left) and 35S::GFP (Right) transgenic plants grown in 100 μmol m-2·s-1 white light and illuminated with 3 μmol m-2·s-1 UV-B for 4 h: lane 1, DNA immunoprecipitated by using anti-GFP antibody; lane 2, no antibody control; lane 3, input DNA before immunoprecipitation; lane 4, PCR without added DNA.

Tables

  • Table 1. UVR8 and HY5 regulate a range of genes concerned with UV protection
    wt UV-B versus uvr8 UV-B
    		wt UV-B versus hy5 UV-B
    Probe-set ID Gene Name RPscore FDR FCrma RPscore FDR FCrma
    258321_at At3g22840 Early light-induced protein (ELIP1) 1.7 0 -156.71 40.27 0 -5
    245306_at At4g14690 Early light-induced protein (ELIP2) 2.36 0 -92.41 279.78 1.77 -2.59
    252123_at At3g51240 Flavanone 3-hydroxylase (F3H) 3.26 0 -63.22 46.69 0 -4.71
    253496_at At4g31870 Glutathione peroxidase, putative 4.71 0 -43.67
    250207_at At5g13930 Chalcone synthase (CHS) 5.5 0 -34.76 5.08 0 -9.96
    249063_at At5g44110 ABC transporter family protein 6.42 0 -28.16 230.02 1.22 -2.86
    250533_at At5g08640 Flavonol synthase 1 (FLS1) 6.85 0 -26.52 2.35 0 -13.32
    249769_at At5g24120 RNA polymerase subunit SigE (sigE) 9.6 0 -20.23
    251020_at At5g02270 ABC transporter family protein 9.96 0 -20.08 401.58 3.14 -2.45
    247463_at At5g62210 Embryo-specific protein-related 11.13 0 -19.05 1.66 0 -15.7
    246966_at At5g24850 Cryptochrome dash (CRYD) 13.66 0 -15.39 5.23 0 -9.7
    245560_at At4g15480 UDP-glucoronosyl/UDP-glucosyl transferase family protein 15.21 0 -14.56 18.61 0 -6.33
    251658_at At3g57020 Strictosidine synthase family protein 16.73 0.07 -13.45
    252010_at At3g52740 Expressed protein 21.41 0.07 -11.51 323.82 2.37 -2.66
    251827_at At3g55120 Chalcone isomerase (CHI) 23.11 0.06 -10.84 20.88 0 -6.08
    249191_at At5g42760 O-methyltransferase N terminus domain-containing protein 23.81 0.06 -11.05
    251727_at At3g56290 Expressed protein 24.83 0.06 -10.23
    253039_at At4g37760 Squalene monooxygenase, putative 25.89 0.05 -10.12
    246468_at At5g17050 Flavonoid 3-O-glucosyl transferase 26.33 0.05 -9.76
    250420_at At5g11260 bZIP protein HY5 (HY5) 27.24 0.05 -9.93 58.93 0.03 -4.56
    248049_at At5g56090 Cytochrome oxidase assembly family protein 30.44 0.05 -9.05 83.53 0.12 -3.67
    250083_at At5g17220 Glutathione S-transferase, AtGST12/TT19 31.93 0.04 -8.64 16.03 0 -6.77
    249215_at At5g42800 Dihydroflavonol 4-reductase (DFR) 32.04 0.04 -8.7 56.27 0.04 -4.52
    263122_at At1g78510 Solanesyl diphosphate synthase (SPS) 33.57 0.04 -8.5
    259537_at At1g12370 Type II CPD photolyase PHR1 (PHR1) 35.67 0.04 -8.03 184.55 0.75 -3.03
    253943_at At4g27030 Expressed protein 36.84 0.04 -8.09
    260955_at At1g06000 UDP-glucoronosyl/UDP-glucosyl transferase family protein 37.27 0.03 -8.12 98.76 0.17 -3.98
    250794_at At5g05270 Chalcone-flavanone isomerase family protein 38.01 0.03 -8.25 15.62 0 -6.55
    249798_at At5g23730 Transducin family protein/WD-40 repeat family protein 38.51 0.03 -7.73 332.09 2.46 -2.5
    262626_at At1g06430 FtsH protease, FtsH8 41.46 0.03 -7.46
    252661_at At3g44450 Expressed protein 42.44 0.03 -7.55
    262705_at At1g16260 Protein kinase family protein 47.38 0.06 -6.86
    253879_s_at At4g27570 Glycosyltransferase family protein 54.76 0.05 -6.35 42.2 0 -4.96
    248347_at At5g52250 Transducin family protein/WD-40 repeat family protein 56 0.05 -6.31
    255594_at At4g01660 ABC1 family protein 56.86 0.05 -6.24
    258349_at At3g17610 bZIP transcription factor HY5-like protein (HYH) 58.07 0.04 -6.01
    265634_at At2g25530 AFG1-like ATPase family protein 61.67 0.09 -5.95 185.64 0.74 -2.96
    260072_at At1g73650 Expressed protein 66.05 0.1 -5.56
    264752_at At1g23010 Multi-copper oxidase type I family protein 69.4 0.1 -5.7 104.77 0.2 -3.65

    • The genes listed are induced by UV-B in wild type but show much reduced UV-B induction in uvr8-1 (left columns) and, in many cases, hy5-1 (right columns). The data-set comparisons used for gene identification are explained in Materials and Methods, and the complete data for 72 genes are presented in Table 2, which is published as supporting information on the PNAS web site. The microarray data were analyzed by the Rank Products method (22); RPscore is a measure of differential expression, FDR indicates the expected percentage of false positives, and FCrma is a measure of the fold change in expression. The Probe-set ID is the Affymetrix probe-set identifier.

Data supplements

  • Brown et al. 10.1073/pnas.0507187102.

    Supporting Table

    Files in this Data Supplement:

    Supporting Table 2




    Supporting Table 2

    Table 2. Genes regulated by UVR8 and HY5.

    Listed are 72 genes induced in response to UV-B in wild-type Arabidopsis that showed much-reduced UV-B induction in the uvr8-1 mutant. Further indicated are which of the UVR8-regulated genes are also regulated by HY5.

    The UVR8-regulated genes were identified in the following procedure. First, the comparison of transcripts in wild-type exposed to UV-B versus uvr8-1 exposed to UV-B, cut at 5% false discovery rate (FDR) (yellow), identified genes with reduced expression in the mutant. Second, each of the genes listed was verified as UV-B induced in wild type by comparison of wild type exposed to UV-B versus wild type in low fluence rate white light (LW), cut at 5% FDR (cyan), but at the same time not identified as elevated in expression in uvr8-1 exposed to UV-B versus uvr8-1 in low fluence rate white light, at 5% FDR (green). Genes regulated by HY5 were identified through comparison of wild-type exposed to UV-B and hy5 exposed to UV-B; values are shown only for genes with FDR <5% (tan).

    The data are derived from Affymetrix whole-genome microarray analyses of triplicate RNA samples. Microarray preparation and data analysis were carried out as described in Materials and Methods. The Probe-set ID is the Affymetrix probe-set identifier. RPscore is a measure of differential expression calculated as geometric mean of the gene ranks in all between-chip comparisons contributing to a given between-group comparison by the rank product method (1). FDR is an estimate of False Discovery Rate indicating the expected percentage of false positives in the list sorted by RPscore and cut at the particular transcript. FDR linked to a given transcript shows the minimum FDR at which this gene appears as differentially expressed. The FCrma is a fold change calculated as an antilog of a mean log fold change over all possible between-chip comparisons contributing to a given between-group comparison after RMA normalisation (2). The FCnom corresponds to more realistic fold-change values obtained from the FCrma using a correction procedure to compensate for RMA-specific distortion of fold-change values (3). Gene is the Arabidopsis gene identifier. Symbol is a gene common name. Title is a brief gene annotation. The values described as NA mean that the gene in question was not identified as differentially expressed at the FDR cutoff of 50%.

    1. Breitling, R., Armengaud, P., Amtmann, A. & Herzyk, P. (2004) FEBS Lett. 573, 83-92.

    2. Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B & Speed, T. P. (2003) Nucleic Acids Res. 31, e15.

    1. Cope, L. M., Irizarry, R. A., Jaffee, H.A., Wu, Z. & Speed, T.P. (2004) Bioinformatics 20, 323-331.
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A UV-B-specific signaling component orchestrates plant UV protection
Bobby A. Brown, Catherine Cloix, Guang Huai Jiang, Eirini Kaiserli, Pawel Herzyk, Daniel J. Kliebenstein, Gareth I. Jenkins
Proceedings of the National Academy of Sciences Dec 2005, 102 (50) 18225-18230; DOI: 10.1073/pnas.0507187102
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