Photoreceptor-Mediated Bending towards UV-B in Arabidopsis

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Photoreceptor-Mediated Bending towards UV-B in Arabidopsis Vandenbussche Filip , Tilbrook Kimberley , Fierro Ana Carolina , Marchal Kathleen , Poelman Dirk , Van Der Straeten Dominique , Ulm Roman   Molecular Plant  Volume 7, Issue 6, Pages 1041-1052 (June 2014) DOI: 10.1093/mp/ssu039 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Unilateral Irradiation Stimulates Bending towards UV-B in Wild-Type, phot1 phot2 and nph3. (A) Wild-type (Col), phot1 phot2, and nph3 were grown for 2 d in darkness on vertical agar plates and subsequently exposed to unilateral monochromatic UV-B (302nm) or blue light (450nm) (0.12 μmol m–2 s–1) for 24h. Error bars: SD (n > 10). (B) Two-day-old etiolated seedlings were exposed to unilateral white or narrowband UV-B for 3 d and photographed. (C) Kinetics of bending towards UV-B. Angle of curvature towards unilateral monochromatic UV-B in 2-day-old etiolated wild-type (Col) and phot1 phot2 seedlings. UV-B (0.12 μmol m–2 s–1) exposure started at time = 0min. Error bars: SD (n = 10). Molecular Plant 2014 7, 1041-1052DOI: (10.1093/mp/ssu039) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 UVR8 Mediates Bending towards UV-B in the Absence of Phototropins. (A) Immunoblot analysis of UVR8 and actin (loading control) levels in 3-day-old seedlings (4 d after imbibition), comparing three independent phot1 phot2 amiR-uvr8 lines (T3 generation) with wild-type (Col) and phot1 phot2. (B) Quantitative RT–PCR analysis of HY5 mRNA levels in response to UV-B in 3-day-old phot1 phot2 amiR-uvr8 transgenic seedlings compared to wild-type (Col) and phot1 phot2 controls. Error bars represent SE of the technical triplicates. CNRQ, calibrated normalized relative quantity. (C) Phototropic response of wild-type (Col), uvr8, phot1 phot2, and phot1 phot2 amiR-uvr8 line #5 to unilateral UV-B. Two-day-old etiolated seedlings were exposed for 3 d to unilateral narrowband UV-B and photographed. Arrows indicate the direction of incoming UV-B irradiation. (D) Seedlings were grown for 2 d in the dark, and subsequently exposed for 18h to unilateral monochromatic UV-B (306nm, 0.12 μmol m–2 s–1). Molecular Plant 2014 7, 1041-1052DOI: (10.1093/mp/ssu039) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Directional Bending towards UV-B Involves Auxin. (A) Quantification of epidermal staining on the illuminated and shaded sides of hypocotyls of DR5rev::GFP seedlings in wild-type (‘DR5’) and phot1 phot2 (‘phot1phot2DR5’) background exposed for 18 h to unilateral UV-B (302nm) or blue (450nm) light (each 0.12 μmol m–2 s–1). (B) Confocal microscopy image of GFP localization in the bending zone of phot1 phot2/DR5rev::GFP phototropic hypocotyls. Seedlings were grown for 2 d in the dark then sujected to 18h of unidirectional UV-B (0.12 μmol m–2 s–1). (C) Phototropic bending response of wild-type, phototropin, and auxin transport mutants. Seedlings were grown and treated as for (B). NPA: Col-0 was treated from germination on with 20 μM of NPA. Error bars: SD (n > 10). Molecular Plant 2014 7, 1041-1052DOI: (10.1093/mp/ssu039) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Repression of Auxin-Responsive Genes by UV-B Is Mediated by UVR8. (A) Heat map of genes down-regulated by UV-B, compared with their auxin responsiveness. Red indicates induction, blue indicates repression, and white indicates no difference in expression. ‘627’ and ‘863’ refer to the numbers of the experiments in the ArrayExpress database (see the ‘Methods’ section). WT, wild-type; hyp, isolated hypocotyls. The gray column on the left indicates genes that show statistically significant down-regulation by UV-B and up-regulation by auxins. AGI, Arabidopsis Genome Initiative identifier. Corresponding gene lists can be found in Supplemental Tables 1–4. (B) Quantitative RT–PCR analysis of SAUR27, IAA19, IAA29, and SAUR23 mRNA levels in response to UV-B in 3-day-old uvr8 mutant seedlings compared to wild-type (Col). Error bars represent SE of triplicate biological replicates. Expression levels are depicted relative to those Col 0h UV-B samples. Black bars: 0 h UV-B; light gray bars: 1 h UV-B; dark-gray bars: 6 h UV-B. (C) Phototropic bending response of wild-type, phototropin, and auxin signaling mutants. Seedlings were grown for 2 d in the dark then subjected to 18h of unidirectional UV-B (302nm) or blue (450nm) (each 0.12 μmol m–2 s–1). Error bars: SD (n > 10). Asterisks indicate statistically significant differences from the wild-type (Col-0) control in the same condition (p ≤ 0.05). Molecular Plant 2014 7, 1041-1052DOI: (10.1093/mp/ssu039) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Working Model of the UVR8-Mediated Bending Response towards UV-B. When an elongating stem is exposed to unidirectional UV-B, UVR8 is activated and monomerizes mainly on the illuminated side of the stem. UVR8 signaling leads to down-regulation of the auxin response (manifested as repression of auxin-responsive genes) and efflux may be stimulated. Consequently, elongation is reduced. On the shaded side, UVR8 remains less activated and mainly in its dimer form, allowing elongation to continue normally. Differential elongation rates on either side of the stem then leads to a directional growth response. Note: the contribution of phototropins in UV-B-mediated phototropism of etiolated seedlings is not included in the working model. Molecular Plant 2014 7, 1041-1052DOI: (10.1093/mp/ssu039) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions