Fig 1.
UVR8-dependent stabilization of HFR1 suppresses activation of shade marker genes.
(A) Anti-HA immunoblot analysis of HFR1-HA levels in 7-day-old Col/ProHFR1:HFR1-3xHA (HFR1-HA) and uvr8-6/ProHFR1:HFR1-3xHA (uvr8/HFR1-HA) seedlings grown in long-day conditions under white light (WL) or white light supplemented at ZT3 with 3-h UV-B (+UVB), low R:FR (+FR), or a combination of both (+FR+UVB). Wild type (Col) is shown as negative control. The immunoblot was re-probed with anti-UVR8, as well as anti-actin as loading control. (B–E) RT-qPCR analysis of (B) PIL1, (C) XTR7, (D) ATHB2, and (E) IAA29 expression in 7-day-old wild-type (Col), uvr8-6, and hfr1-101 seedlings grown under white light and exposed to either UV-B (+UVB), low R:FR (+FR), or both (+FR+UVB) for 3 h or maintained under white light (WL). Error bars represent SEM of three biological replicates. Asterisks indicate a significant difference in transcript abundance compared to that under WL in each genotype (* p < 0.05; ** p < 0.01).
Fig 2.
UV-B-activated UVR8 broadly suppresses shade-responsive gene expression.
(A) Pie charts showing the percentage and number of shade-induced genes that are repressed by UV-B (FC > 2, adjusted pvalue < 0.05) in wild type (Col), uvr8-6, and hfr1-101 seedlings. (B) Venn diagram representing the intersection between shade-induced genes in wild type (Col +FR/WL ↑), uvr8-6 (uvr8 +FR/WL↑), and hfr1-101 (hfr1 +FR/WL ↑) seedlings (FC > 2, adjusted pvalue < 0.05). (C) Venn diagram representing the intersection between shade-induced genes that are repressed by UV-B in a UVR8-dependent manner in wild type (Col) but not in hfr1-101 (FC > 2, adjusted pvalue < 0.05). The intersection of 44 genes is referred to as the PIL1 cluster. (D, E) RT-qPCR analysis of (D) ST2A and (E) FLP1 expression in 7-day-old Col, hfr1-101, and hfr1-4 seedlings grown under the indicated light conditions for 3 h. Error bars represent SEM of three biological replicates. Asterisks indicate a significant difference compared to that under WL in each genotype (* p < 0.05; ** p < 0.01).
Fig 3.
PIL1 and HFR1 act redundantly in UVR8-mediated repression of shade-induced FHL and PIL2 expression.
(A, B) RT-qPCR analysis of (A) FHL and (B) PIL2 expression in 7-day-old seedlings of wild type (Col), hfr1-101, pil1-6, and hfr1-101 pil1-6 grown under constant white light (WL) and transferred to the indicated experimental light conditions for 3 h. Error bars represent SEM obtained from three biological replicates. Asterisks indicate the statistical significance compared to WL in each genotype (* p < 0.05; ** p < 0.01).
Fig 4.
UVR8 induces PIF4 and PIF5 degradation in response to UV-B, independently of BOP2.
Anti-HA immunoblot analysis of HA-tagged PIF4 and PIF5 proteins in 7-day-old seedlings grown in long-day conditions under white light and exposed to low R:FR with (+) or without (-) 3-h supplemental narrowband UV-B at ZT3. Wild type (Col) is shown as negative control. (A) PIF4-HA protein levels were analyzed in Col/ProPIF4:PIF4-3xHA (PIF4-HA) and uvr8-6/ProPIF4:PIF4-3xHA (uvr8/PIF4-HA). (B) PIF5-HA protein levels were analyzed in Col/ProPIF5:PIF5-3xHA (PIF5-HA) and uvr8-6/ProPIF5:PIF5-3xHA (uvr8/PIF5-HA). (C) PIF4-HA protein levels were analyzed in pif4-101/ProPIF4:PIF4-3xHA (pif4/PIF4-HA) and pif4-101 bop2-2/ProPIF4:PIF4-3xHA (pif4 bop2/PIF4-HA). (D) PIF4-HA protein levels were analyzed in Col/ProPIF4:PIF4-3xHA (PIF4-HA) and cop1-4/ProPIF4:PIF4-3xHA (cop1-4/PIF4-HA). Blots were re-probed with anti-UVR8 (A and B) as well as anti-actin as loading control (A–D). Numbers under lanes indicate relative band intensities.
Fig 5.
UV-B impairs PIF4 binding to shade marker genes’ promoters in a UVR8-dependent manner.
(A) Schematic representation of PIL1 and XTR7 with G-boxes and regions amplified in ChIP-qPCR indicated. (B, C) PIF4-HA chromatin association in Col/ProPIF4:PIF4-3xHA (PIF4-HA) and uvr8-6/ProPIF4:PIF4-3xHA (uvr8/PIF4-HA). Ten-day-old seedlings were grown in long-day conditions under white light and exposed to low R:FR with (+) or without (-) 3-h supplemental narrowband UV-B at ZT3. ChIP-qPCR was performed for (B) PIL1 and (C) XTR7 promoters. ChIP data are presented as the percentage recovered from the total input DNA (% Input). Data shown are representative of three independent biological replicates (see S5A–S5D Fig). Error bars represent SD of three technical replicates. Immunoprecipitated DNA was quantified by qPCR using primers in the promoter regions containing G-boxes (region 1, ProPIL1_-1417; region 3, ProXTR7_-197) or control regions without G-boxes (region 2, ProPIL1_+1816; region 4, ProXTR7_-664).
Fig 6.
UV-B-induced PIF4 degradation and reduced association with shade marker genes’ promoters is independent of HFR1.
(A) Anti-HA immunoblot analysis of PIF4-HA in 7-day-old seedlings grown in long-day conditions under white light and exposed to 3-h low R:FR (+FR) at ZT3 in the presence (+UVB) or absence of supplemental UV-B (-UVB). Wild type (Col) treated with low R:FR is shown as negative control. PIF4-HA protein levels were analyzed in Col/ProPIF4:PIF4-3xHA (PIF4-HA) and hfr1-101/ProPIF4:PIF4-3xHA (hfr1/PIF4-HA). Blot was re-probed with anti-actin as loading control. (B) Quantification of the immunoblot shown in (A). Error bars represent SD of three biological replicates. (C) Chromatin association of PIF4-HA in 10-day-old Col/ProPIF4:PIF4-3xHA (PIF4-HA) and hfr1-101/ProPIF4:PIF4-3xHA (hfr1/PIF4-HA) seedlings grown in long-day conditions under white light and exposed at ZT3 to 3-h low R:FR with (+) or without (-) supplemental UV-B. Col was included as negative control. ChIP-qPCR was performed for the PIL1 promoter. ChIP of DNA associated with PIF4-HA is presented as the percentage recovered from the total input DNA (% Input). Data shown are representative of two independent biological replicates (see S5E and S5F Fig). Error bars represent SD of three technical replicates. Immunoprecipitated DNA was quantified by qPCR using primers in the promoter region containing a G-box (region 1, ProPIL1_-1417) or control region without a G-box (region 2, ProPIL1_+1816). (D) Schematic representation of PIL1 with G-boxes and regions amplified in ChIP-qPCR indicated.