Volume 7, Issue 5, Pages 773-791 (May 2014) Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B  Fasano Rossella , Gonzalez Nathalie , Tosco Alessandra , Dal Piaz Fabrizio , Docimo Teresa , Serrano Ramon , Grillo Stefania , Leone Antonella , Inzé Dirk   Molecular Plant  Volume 7, Issue 5, Pages 773-791 (May 2014) DOI: 10.1093/mp/ssu002 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Complementation of mpk1 ppz1 Double Mutants by UVR8. The indicated strains, wild-type (W303), mpk1 ppz1double mutant, and mpk1 ppz1 transformed with the vector containing UVR8 cDNA (PFL61-UVR8), were grown on selective medium containing 1M sorbitol (SORB.), as described in the ‘Methods’ section, serially diluted in sterile water plus 1M sorbitol, and spotted onto YPD plates or YPD plates containing 1M sorbitol. Images were taken after 3 d of incubation at 37°C. Similar results were obtained in three separate experiments. Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 UVR8 Expression under Abiotic Stress. (A)UVR8 mRNA expression in plants grown under normal conditions or upon exposure to abiotic stress. UVR8 relative expression levels in 2-week-old plants grown in vitro on MS medium or after transfer for 24h to medium supplemented with NaCl (100mM) or PEG (–0.5MPa); after transfer to starvation conditions (no sugar, dark); or after exposure to 5 μmol m−2 s−1 of UV-B light for 2h. Relative transcript levels of UVR8 were determined by qRT–PCR and the values were normalized to the untreated plants using 18S ribosomal RNA as an internal control. Data represent average of three independent experiments ± SD. (B) UVR8 protein accumulation in wild-type plants after exposure to NaCl (100mM) or PEG (–0.5MPa) for 24h. Plants were grown as reported above and UVR8 protein levels were analyzed by Western blotting with an anti-UVR8 antibody. The analysis was performed in triplicate and UVR8 relative protein levels were determined by densitometric analysis using Image J. The values were normalized to the untreated plants using rubisco large subunity (RBCL) and averages ± SD are shown (n = 3; P < 0.05). Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phenotypic Characterization of 35S-UVR8 Plants. (A) Seven-day-old seedlings overexpressing UVR8 (right) as compared to control plants (left), grown in vitro at 23°C under a 16-h light/8-h dark photoperiod. (B) Thirty-day-old UVR8 transgenic plants (right) grown in soil. At this time, UVR8-overexpressing plants had already produced an inflorescence. (C) Photographs of representative leaf series of 35S-UVR8 plants. Plants were grown in vitro at 23°C under a 16-h light/8-h dark photoperiod and leaf series (cotyledons on the left and youngest leaves on the right) photographed 21 d after sowing (DAS); bar = 10mm. (D) Area of rosette leaves at different positions. Values are averages ± SD (n = 14; * P = 0.05). (E) Six-week-old control (left) and 35S-UVR8 (right) plants grown in soil. (F) First leaves (leaf1–2) of 30-day-old control (left) and 35S-UVR8 (right) plants grown in vitro. (G) Siliques of 35S-UVR8 (right) plants are visibly shorter than those of the control (left). (H) Leaf measurements of 21-day-old 35S-UVR8 plants grown in vitro. To analyze the cellular basis of the size differences between the control and 35S-UVR8, the leaf area, the epidermal cell number, and cell area were measured. Values are averages ± SD (n = 5; * P = 0.05). Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Response of 35S-UVR8 Plants to Severe Stress. (A) Growth analysis on severe stress conditions. Plants were grown in vitro for 2 weeks on meshes placed on MS medium and transferred on MS media supplemented with NaCl (150mM) or mannitol (200mM). Plants were photographed after 3 weeks and the percentage of survival was recorded. The results are averages of two independent replicates (n = 70–80) ± SD. (B) Phenotypes of 35S-UVR8 or control plants grown for 14 d and 28 d on 200mM mannitol under mylar filter used to screen out the UV-B light. Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 CHS expression and Analysis of Flavonoid Content in 35S-UVR8 Plants. (A) Accumulation of CHS mRNA in 7-day-old transgenic UVR8-overexpressing seedlings grown under low levels of UV-B. Relative transcript levels of UVR8 were determined by qRT–PCR and normalized using ACT2 as internal control. Error bars represent standard deviations of three replicates. (B) Levels of flavonols accumulating in leaves of 35S-UVR8 plants compared to control plants. The main flavonol derivatives were kaempferol-3-O-[rhamnosyl (1>2) glucoside]-7-O-rhamnoside (K1), kaempferol-3-O-glucoside-7-O-rhamnoside (K2) and kaempferol-3-O-rhamnoside-7-O-rhamnosiden (K3). The values are expressed as nmol g–1 dry weight. Mean values and standard deviations are given for three replicates. (C) HPLC of soluble phenolics extracted from 35S-UVR8 plants grown in soil for 5 weeks. Profile of UVR8 transgenic extracts compared to control plants, at 345nm. The sinapoyl derivatives were sinapoyl glucose (S1), sinapoylmalate (S2), and disinapoylglucose (S3). (D) Structure of flavonol glycosides and sinapoyl derivatives were inferred from their mass spectral properties. Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Root Phenotypes of 35S-UVR8 Plants. (A) Seven-day-old 35S-UVR8 seedlings (right; control plant on the left) grown in vitro in a growth chamber at 23°C under continuous light. (B) Fourteen-day-old 35S-UVR8 plants (right, control plants on the left) grown in vitro. The arrow head indicates the bottom part of the branching zone of the root. (C) Lateral root distribution in different sections of roots (as indicated in (B)) of 14-day-old 35S-UVR8 plants. Data are averages ± SE (n = 20). (D) Detailed staging of the root. Data are averages (n = 8–12). (E) DPBA-visualized flavonol accumulation in 7-day-old 35S-UVR8 seedlings compared to the control and fluorescence intensity calculated by ImageJ. Data are averages ± SE (n = 12–16). Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Response of uvr8-6 Plants to 150mM NaCl. (A) Response of the uvr8-6 mutant to severe stress under low levels of UV-B. Plants were grown in vitro for 2 weeks and then transferred to MS medium supplemented with NaCl (150mM). Plants were photographed after 3 weeks on stress conditions and the percentage of survival (on the left) and the percentage difference of rosette size upon salt stress (on the right) were recorded. Survival rates and SD were calculated from the results of three independent experiments (n = 80 plants/genotype). (B) Phenotypes and response of the uvr8-6 mutant to severe stress under low levels of UV-B or under mylar filters used to screen out the UV-B light. Plants were grown as above and were photographed after 1 week on stress conditions and the percentage of survival was scored. Data are averages ± SD (n = 90). Molecular Plant 2014 7, 773-791DOI: (10.1093/mp/ssu002) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions