1. Phytochrome A Negatively Regulates the Shade Avoidance Response by Increasing Auxin/Indole Acidic Acid Protein Stability 
Chuanwei Yang, Famin Xie, Yupei Jiang, Zhen Li, Xu Huang, Lin Li 
Developmental Cell 
Volume 44, Issue 1, Pages e4 (January 2018)
DOI: /j.devcel
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2. Figure 1
Effects of Shade Treatments on Hypocotyl Elongation, Protein Level, and Localization of PHYA
(A) Hypocotyl length of wild-type and phyA under different shade conditions. Seedlings were germinated and grown for 5 days under white light and either kept in white light or transferred to different shade (0.8, 0.4, and 0.2) for 4 days. Data are presented as the means ± SD. More than 20 seedlings were measured. See also Figure S1.
(B) Immunoblot of PHYA using anti-PHYA antibody in white-light-grown Col-0 seedlings, which were transferred to different shade (0.2, 0.4, and 0.8) for indicated times. Histone H3 indicates loading control. See also Figure S2A.
(C) Immunoblot of PHYA-YFP using anti-GFP antibody in white-light-grown 35S:: PHYA-YFP/phyA-211 transgenic seedlings transferred to different shade (0.2, 0.4, and 0.8) for 2, 4, 8, and 12 hr. Histone H3 indicates loading control. See also Figure S2C.
(D) Localization of PHYA-YFP on top of hypocotyl of transgenic seedlings under different light conditions. Five days white-light-grown 35S::PHYA-YFP/phyA-211 seedlings were transferred to different shade (0.2, 0.4, and 0.8) for 2 and 8 hr. Images of the YFP signal were obtained using confocal microscopy. White scale bar represents 25 μm. See also Figure S2F.
(E) Shade upregulates PHYA abundance more in the nuclear fraction. Immunoblot of PHYA-YFP proteins using anti-GFP antibody in the total, nuclear, and non-nuclear fractions from white-light- and shade-treated seedlings. Histone H3 is a nuclear marker, UGPase (UDP-glucose pyrophosphorylase) is a cytoplasm marker, and RubisCO large subunit (RbcL) is a chloroplast protein, as a non-nuclear fraction marker.
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3. Figure 2
Expression Level of Auxin-Related Genes in phyA-211 under Different Shade Conditions
(A) Heatmap of GO descriptor enrichment in Col-0 and phyA-211 upon exposure to two shade conditions. Heatmap displays the p value of enriched GO descriptors by means of DAVID in wild-type and phyA-211 seedlings for two shade conditions. See also Tables S2 and S4.
(B) Heatmap represents the relative expression level of auxin-responsive genes. Red, white, and blue rows indicate RNA expression in high, medium, and low levels, respectively. Boxplot displays expression of auxin-responsive genes in Col-0 and phyA-211. FPKMs, fragments per kilobase of exon per million fragments mapped. The p value presents the significant difference between two genotypes. The paired t test was used for the statistical analysis. See also Table S5.
(C) Relative gene expression of several auxin-responsive genes (HAT2, IAA5, GH3.3, GH3.5, and SAUR66) in Col-0 and phyA-211 under white light and two shade conditions by qRT-PCR. Data are presented as the means ± SD of three biological replicates.
(D) Heatmap represents the relative expression level of auxin metabolism-related genes in Col-0 and phyA-211 under two shade conditions. Boxplot displays the expression of auxin metabolism-related genes in two genotypes. The p value presents the significant difference between two genotypes. See also Figure S3C and Table S5.
(E) Free IAA contents in Col-0 and phyA-211 under white light and two shade conditions. Col-0 and phyA-211 were grown in white light and kept in white light or transferred to shade for 1 hr. Aerial parts were collected to measure the free IAA level. Data are presented as the means ± SD of two biological replicates.
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4. Figure 3 Sensitivity of phyA Mutants to Application of Auxin
(A) Phenotype of wild-type and phyA mutant seedlings treated with NPA, yucasin, and IAA (indole-3-acetic acid) grown under shade (0.2) conditions. Seedlings were grown in liquid 1/2 MS medium containing NPA, yucasin, and IAA (0, 1 μM, 10 μM, and 50 μM) under white-light conditions for 5 days and kept in white light or transferred to shade (0.2) for 4 days before the hypocotyl measurement. Data are presented as the means ± SD. At least 20 seedlings were measured. White scale bar represents 2 mm.
(B) Fold change of hypocotyl length of seedlings grown under shade (0.2) with IAA compared with seedlings without IAA treatment. Each shape indicates independent replication of the measurement. ∗p < 0.05, ∗∗p < 0.01 by paired t test versus their wild-type controls.
(C) Phenotype of wild-type and phyA mutant seedlings treated with NPA, yucasin, and IAA grown under weak white light (∼10 μmol·m−2·s−1). Data are presented as the means ± SD. At least 20 seedlings were measured. White scale bar represents 2 mm.
(D) Fold change of hypocotyl length of seedlings grown under weak white light (∼10 μmol·m−2·s−1) with IAA compared with seedlings without IAA treatment. Each shape indicates independent replication of the measurements. ∗p < 0.05, ∗∗p < 0.01 by paired t test versus their wild-type controls.
(E) Auxin induction of HAT2, IAA5, IAA29, SAUR62, and SAUR66 gene expression in wild-type and phyA-211. Five-day weak white-light-grown seedlings in liquid 1/2 MS medium containing NPA and yucasin were treated with 1.5 μM IAA for 1 hr. Data are presented as the means ± SD of three biological replicates.
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5. Figure 4 Interaction between PHYA and AUX/IAAs
(A) Interaction between PHYA and AUX/IAAs was detected with bimolecular fluorescence complementation assay based on firefly luciferase (LUC). N-terminal and C-terminal halves of LUC were fused to AUX/IAAs and PHYA, respectively. Constructs were co-expressed in tobacco leaf cells. Luciferin was infiltrated before LUC activity was monitored.
(B–F) Top panel, PHYA-YFP in the pull-down fractions and inputs was analyzed by western blot using anti-GFP antibody. Bottom panel, Coomassie brilliant blue-stained proteins on the SDS-PAGE gel (CBS) are shown.
(B) Interaction between PHYA and AUX/IAAs was detected by semi-in vivo pull-down assay. AUX/IAAs fused with GST were expressed and purified from E. coli. Protein extracts from plants overexpressing PHYA-YFP (35S::PHYA-YFP/phyA-211) after 12 hr shade (0.8 and 0.2) treatment.
(C) GST-IAAs pulled down PHYA extracted from dark- and FR-treated 35S::PHYA-YFP/phyA-211 seedlings. Protein extracted from plants overexpressing PHYA-YFP (35S::PHYA-YFP/phyA-211) grown in the dark for 4 days and maintained in the dark or transferred to FR (∼20 μmol·m−2·s−1) for 1 hr.
(D) GST-IAAs pulled down PHYA extracted from dark- and R-treated 35S::PHYA-YFP/phyA-211 seedlings. Protein extracted from plants overexpressing PHYA-YFP (35S::PHYA-YFP/phyA-211) grown in the dark for 4 days and maintained in the dark or transferred to R (∼20 μmol·m−2·s−1) for 1 hr with MG132 (+) or without MG132 (−).
(E) IAA3 I/II (1–77 aa) and IAA17 I/II (1–95 aa), not IAA3 III/IV (78–112 aa) and IAA 17 III/IV (96–229 aa), could pull down PHYA-YFP.
(F) PHYA ΔN100 (deletion of 100 amino acids from the N terminus of PHYA) expressed in tobacco leaf cells can be pulled down by GST-IAA3/17, but PHYA ΔC310 (812 amino acids from the N terminus of PHYA, deletion of 310 amino acids from the C terminus of PHYA) expressed in tobacco leaf cells could not be pulled down by GST-IAA3/17.
(G) Interaction of PHYA and AUX/IAAs in tobacco leaf cells detected by coimmunoprecipitation (coIP). Two constructs were co-expressed in tobacco leaf cells. Anti-GFP Sepharose beads were used to precipitate PHYA-YFP. Western blot was probed with anti-LUC antibody for immunoprecipitated samples.
(H) Interaction of PHYA and AUX/IAAs in Arabidopsis detected by coIP. Anti-GFP Sepharose beads were used to precipitate PHYA-YFP from plants overexpressing both PHYA-YFP and IAA17-LUC or IAA17-LUC grown under white light or after 1 hr shade (0.2) treatment. Seedlings were treated with 100 μM MG132 for 1 hr before light treatment. Western blot was probed with anti-LUC antibody for immunoprecipitated samples.
(I) PHYA-YFP interacts with IAA17-LUC in the nuclear fraction. Immunoblot of IAA17-LUC using anti-LUC and PHYA-YFP using anti-GFP in the total, nuclear, and non-nuclear fractions from dark-grown PHYA-YFP/IAA17-LUC seedlings treated with 1 hr R or FR. MG132 was used to maintain protein abundance. Histone H3 is a nuclear marker and UGPase is a cytoplasm marker.
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6. Figure 5
Degradation of IAA17 Fused with LUC in phyA-211 and Col-0 under White Light and Shade
(A) LUC activity of IAA17-LUC measured in over 45 seedlings in phyA-211 and Col-0. The same seedlings grown under white-light conditions were maintained in white light or transferred to shade (0.8, 0.4 and 0.2) for 1 hr. LUC activities were measured before and after light treatments.
(B) Relative fold change of LUC activity of IAA17-LUC in phyA-211 and Col-0 background by 1 hr white-light or shade treatment. Calculation based on data presented in (A). Significant differences between phyA-211 and Col-0 are shown as asterisks. Data are presented as the means ± SD (n > 45). ∗∗∗p < by t test.
(C) Images of the fluorescence signal of IAA17-LUC in phyA-211 and Col-0 by chemiluminescence detector. The same seedlings grown under white light were maintained in white light or transferred to shade (0.2) for 1 hr.
(D) Immunoblot of IAA17-LUC protein in phyA-211 and Col-0 background. Seedlings were grown in white-light conditions and treated with 1 hr shade (0.8, 0.4, and 0.2). Western blot was probed with anti-LUC antibody. Histone H3 and UGPase indicate loading control.
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7. Figure 6
Effect of PHYA on Degradation of AUX/IAAs Fused with LUC Treated by Auxin
(A) LUC activity of IAA17-LUC was measured in over 50 seedlings in phyA-211 and Col-0 treated by DMSO and 1.5 μM IAA for 5 min. Seedlings were incubated in liquid 1/2 MS with 2 μM NPA and 50 μM yucasin under weak white light (∼10 μmol m−2 s−1). Half of the seedlings were treated with DMSO and half were treated with hormone.
(B) Relative fold change of LUC activity of IAA17-LUC in phyA-211 and Col-0 background by DMSO and 1.5 μM IAA treatment. Calculation based on data presented in (A). Significant differences between phyA-211 and Col-0 are shown as asterisks. Data are presented as the means ± SD (n > 50). ∗p < 0.05 by t test.
(C) Immunoblot of IAA17-LUC protein in phyA-211 and Col-0 background. Seedlings grown in weak white-light conditions with NPA and yucasin and treated with 1.5 μM IAA. Samples were collected at indicated time points. Histone H3 indicates loading control.
(D) PHYA stabilized IAA1-LUC, IAA3-LUC, IAA7-LUC, and IAA17-LUC in tobacco leaf cells treated by auxin. Agrobacterium harboring IAAs-LUC, TIR1-HA, and PHYA-YFP/GFP were co-injected into tobacco leaf cells. A tobacco leaf disc (4 mm diameter) was punched to measure LUC activity before or after 1.5 μM IAA treatments at indicated time points. Data are presented as the means ± SD (n > 6). Significant differences between GFP and PHYA-GFP are shown as asterisks. ∗∗p < 0.01, ∗∗∗p < by t test. See also Figure S5.
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8. Figure 7 PHYA Competes with TIR1 to Bind AUX/IAAs
(A) Abundance of TIR1 under different light conditions. Top panel, 5-day old white-light-grown TIR1-Flag transgenic plants were transferred to shade (0.8, 0.4, and 0.2) for indicated times. Bottom panel, 4-day dark-grown TIR1-Flag transgenic plants were transferred to white light, red light (20 μmol m−2 s−1), or far-red light (20 μmol m−2 s−1) for indicated times. Histone H3 level is shown as loading control.
(B) PHYA-YFP extracted from shade (0.2)-treated samples could compete with TIR1 to bind with IAA3 and IAA17 by semi-in vivo pull-down assay. Recombinant proteins of GST-IAA3/IAA17 were purified from E. coli. TIR1-Flag were extracted from TIR1 overexpression lines. PHYA-YFP was extracted from white-light or shade (0.2)-treated PHYA overexpression seedlings. Anti-Flag and anti-GFP were used to detect the level of TIR1-Flag and PHYA-YFP. GST-IAAs stained with Coomassie brilliant blue are shown at the bottom. See also Figure S7.
(C) Extraction from FR-treated Col-0, but not extraction from FR-treated phyA-211, competed with TIR1 to bind with IAA3/17 in a dose-dependent manner. Dark-grown Col-0 and phyA-211 seedlings were collected after 1 hr FR (∼20 μmol m−2 s−1) treatment. Amount of extract from Col-0 or phyA-211 adjusted according to volume. Anti-Flag and anti-PHYA were used to detect the level of TIR1-Flag and PHYA. GST and GST-IAAs stained with Coomassie brilliant blue are shown at the bottom.
(D) Phenotype of Col-0, phyA-211, axr3-3, and double mutant under white light and shade. Seedlings were grown under white light for 5 days and maintained in white light or transferred to shade for 4 days before the hypocotyl measurement. Data are presented as the means ± SD. At least 20 seedlings were measured.
(E) Proposed model illustrating the role of PHYA on regulating shade-induced hypocotyl elongation. Under shade, dephosphorylated PIF7 promotes auxin biosynthesis and hypocotyl elongation. Under deep shade, accumulated PHYA prevents degradation of AUX/IAAs and suppresses shade-induced hypocotyl elongation.
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