As PIF4 levels are also reduced in mutants and no additional UV-B-mediated degradation is detectable in the absence of functional COP1, COP1-mediated stabilization of PIF4 may be similarly disrupted by activated UVR8, as previously suggested for PIF5 [32]. manifestation in 7-day-old wild-type (Col), seedlings cultivated under white light (WL) and exposed to supplemental 3-h UV-B (+UVB), low R:FR (+FR), or low R:FR and UV-B (+FR+UVB) compared to seedlings taken care of under WL as control. Error bars symbolize SEM of three self-employed biological replicates. Asterisks show a significant difference in transcript large quantity compared to that of WL in each genotype (* p 0.05; ** p 0.01).(PDF) pgen.1008797.s003.pdf (88K) GUID:?23533452-0295-4291-9966-F7853A0DDBA3 S4 Fig: UV-B represses PIF5 binding to promoters of shade marker genes. (A, B) PIF5-HA chromatin association in Col/ProPIF5:PIF5-3xHA (PIF5-HA). Ten-day-old seedlings were cultivated in long-day conditions under white light and revealed at ZT3 to 3-h low R:FR with or without supplemental narrowband UVB. ChIP-qPCR was performed for (A) and (B) promoters. The numbers of the analyzed DNA fragments show the positions of the 5 foundation pair of the amplicon relative to the translation start site (referred to as position Rabbit Polyclonal to DRP1 +1). Fragments designated as ProPIL1_-1417 and ProHFR1_-1689 contain a G-box, whereas ProPIL1_+1816 and ProHFR1_-202 are devoid of a G-box. ChIP of DNA associated with PIF5-HA is definitely offered as the percentage recovered from the total input DNA (% Input). Data demonstrated are representative of three self-employed biological replicates. Error bars symbolize SD of three technical replicates.(PDF) pgen.1008797.s004.pdf (114K) GUID:?9EB77211-4827-4401-B473-D7273F1C2B02 S5 Fig: Self-employed repetitions of ChIP data related to Fig 5B and 5C and Fig 6C. (A-D) PIF4-HA chromatin association in Col/ProPIF4:PIF4-3xHA (PIF4-HA) and (repetitions of data shown in Fig 5B) and (C,D) promoters (repetitions of data shown in Fig 5C). (E,F) Chromatin association of PIF4-HA in 10-day-old Col/ProPIF4:PIF4-3xHA (PIF4-HA) and promoter (repetitions of data demonstrated in Fig 6C). Error bars symbolize SD of three technical replicates.(PDF) pgen.1008797.s005.pdf (81K) GUID:?49AFB17D-AC2A-45F2-BC44-1803FA8AA255 S1 Table: Intersection of genes upregulated by low R:FR and repressed ABT-199 (Venetoclax) by UVB in wild type (Col, S1A Table), (S1B Table) and (S1C Table) (lists corresponding to Fig 2A).(XLSX) pgen.1008797.s006.xlsx (164K) GUID:?09FE5D90-5609-41E7-9BD1-90F73F98BED4 S2 Table: Genes upregulated by low R:FR in Col wild type, (lists corresponding to Fig 2B). (XLSX) pgen.1008797.s007.xlsx (157K) GUID:?66E88F8F-DE2C-4CFD-85EB-EC6155492DB6 S3 Table: Intersection of genes upregulated by low R:FR and repressed by UV-B in wild type and genes upregulated by low R:FR and not repressed by UV-B in mutant (lists corresponding to Fig 2C). (XLSX) pgen.1008797.s008.xlsx (131K) GUID:?B0086379-843E-4549-BEFB-92B12A5593EC S4 Table: Oligonucleotide sequences used in this study. (XLSX) pgen.1008797.s009.xlsx (13K) GUID:?9DAE7DE2-BDF9-423D-94FB-F8D457A00BBA Attachment: Submitted filename: promoter-driven HFR1-HA protein levels in an null mutant background compared to inside a wild-type background. Seven-day-old seedlings were irradiated at Zeitgeber time ZT3 with 3-h supplemental UV-B (+UVB), 3-h supplemental FR to produce low R:FR (+FR), or a combination of the two treatments (+UVB+FR), and compared to seedlings managed in white light as control (WL; -UVB-FR). Low R:FR treatment slightly improved HFR1-HA protein level, both in the absence and presence of UVR8, whereas HFR1 exhibited higher stabilization under UV-B which was UVR8 dependent (Fig 1A). Moreover, UV-B and ABT-199 (Venetoclax) low R:FR stabilized HFR1 in an additive manner (Fig 1A). HFR1 build up was associated with only a fragile transcriptional activation of under UV-B (S1 Fig). These results indicate that active UVR8 signaling prospects to post-translational HFR1 stabilization, likely through COP1 inhibition. Open in a separate windowpane Fig 1 UVR8-dependent stabilization of HFR1 suppresses activation of color marker genes.(A) Anti-HA immunoblot analysis of HFR1-HA levels in 7-day-old Col/ProHFR1:HFR1-3xHA (HFR1-HA) and expression in 7-day-old wild-type (Col), seedlings cultivated less than white light and exposed to either UV-B (+UVB), low R:FR (+FR), or both (+FR+UVB) for 3 h or taken care of less than white light (WL). Error bars symbolize SEM of three biological replicates. Asterisks show a significant difference in transcript large quantity compared to that under WL in each genotype (* p 0.05; ** p 0.01). To test the contribution of HFR1 to UVR8-mediated suppression of low R:FR response, we analyzed ABT-199 (Venetoclax) shade-induced manifestation of color marker genes (((and mutants in comparison to that in crazy type. In each genotype, all four tested genes showed induced manifestation in response to low R:FR (+FR, color), which was strongly suppressed by supplemental UV-B (+FR+UVB).
As PIF4 levels are also reduced in mutants and no additional UV-B-mediated degradation is detectable in the absence of functional COP1, COP1-mediated stabilization of PIF4 may be similarly disrupted by activated UVR8, as previously suggested for PIF5 [32]
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