1. BZR1 Interacts with HY5 to Mediate Brassinosteroid- and Light-Regulated Cotyledon Opening in Arabidopsis in Darkness 
Qian-Feng Li, Jun-Xian He 
Molecular Plant 
Volume 9, Issue 1, Pages (January 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
The bzr1-1D and hy5-215 Mutants Are Insensitive to BR Deficiency-Induced Cotyledon Opening.
(A) The cotyledon phenotypes of the BR-related mutants det2, bri1-5, bin2-1, and bzr1-1D and their respective wild-types (Col-0 and WS) in the dark.
(B) Treatment with 1 μM BRZ induced the opening of the cotyledons of Col-0, but not of the BZR1-dominant mutant bzr1-1D.
(C) bzr1-1D could suppress the cotyledon opening phenotype of the BR-insensitive mutant bri1-5 in the dark.
(D) BRZ responses of the light-related mutants phyA-211, phyB-9, phyA-211 phyB-9, and hy Only the hy5-215 mutant is insensitive to BRZ-induced cotyledon opening in the dark.
(E) hyh, a knock-out mutant of HYH (the close homologue of HY5), responds differently to BRZ in cotyledon opening compared with the hy5-215 mutant.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
The hy5-215 Mutation Suppresses the Cotyledon Opening Phenotype of BR-Insensitive Mutants under BR-Deficient Conditions.
(A) Dynamic changes in cotyledon-opening phenotypes of hy5-215 and bzr1-1D during BRZ treatment at different stages of seedling development. DAG, days after germination.
(B) Cotyledon phenotypes of hy5-215 and its wild-type Col-0, bri1-5 and its wild-type WS, and the hy5-215 bri1-5 double mutant.
(C) Cotyledon phenotypes of hy5-215, bin2-1 and the wild-type Col-0, and hy5-215 bin2-1 double mutant.
(D) Cotyledon phenotypes of bzr1-1D, hy5-215, and the wild-type Col, and the bzr1-1D hy5-215 double mutant.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
BR Has No Effect on the Accumulation of HY5 but Light Can Regulate the Phosphorylation Status and Protein Abundance of BZR1.
(A) Transcript abundance of HY5 in response to BL and BRZ treatment. The BR biosynthetic gene DWF4 was used as a reference for BR treatment.
(B) Transcript abundance of DWF4 and HY5 in the bri1-5 mutant.
(C) Effect of BL and BRZ on the accumulation of HY5-YFP protein.
(D) Transcript levels of BZR1 and the BR biosynthetic genes CPD and DWF4 in response to light and dark treatments.
(E) Transcription of BZR1 in the hy5-215 mutant in the light. 10-day-old Col and hy5-215 seedlings grown in a light–dark cycle (16 h light/8 h dark at 22°C) were collected for qRT–PCR analysis at the end of an 8-h dark period (0 h) and then underwent light treatment for 6 and 12 h, respectively.
(F) Effects of light and dark treatments on the accumulation of the BZR1-CFP protein. The numbers under the blots are the normalized relative protein abundance of phosphorylated (pBZR1) or unphosphorylated BZR1 (BZR1) compared with that of pBZR1 in the first sample, which was set to 1. Tubulin was used as a loading control. L, light; D, dark.
In (A, B, D and E), data are shown as the mean ± standard error SD. Three biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: *P < 0.05; **P < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 BZR1 Interacts with HY5 In Vitro and In Vivo.
(A) Yeast two-hybrid assay for interaction between BZR1 and HY5 proteins.
(B) In vitro pull-down assay for the interaction between full-length HY5 and different versions of BZR1, including the full-length, N-terminal part, and C-terminal part of BZR1.
(C) In vitro pull-down assay for the interaction between the full-length BZR1 and different versions of HY5, including the full-length, N-terminal part, and C-terminal part of HY5.
(D) Co-localization assay of BZR1-GFP and RFP-HY5 in tobacco epidermal cells. The scale bar represents 5 μm.
(E) BiFC analysis of BZR1 and HY5 interaction in planta. Vectors containing the indicated constructs were co-transformed into Nicotiana benthamiana leaves. Both YFP fluorescence images (upper panel) and fluorescence images merged with light view images (lower panel) are shown. The scale bar represents 20 μm.
(F) Co-immunoprecipitation assay of BZR1 with HY5. Total proteins extracted from transgenic plants co-expressing BZR1-GFP and HY5-Myc or BZR1-GFP and the empty Myc vector (Myc) or HY5-Myc and the empty GFP vector (GFP) were immunoprecipitated (IP) by c-Myc antibody-conjugated agarose beads. The precipitated proteins were detected with either an antibody recognizing GFP (anti-GFP) or an antibody recognizing Myc (anti-Myc). The asterisk denotes a non-specific band.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
Ectopic Expression of HY5 Reduces BZR1 Abundance, and HY5 Specifically Binds to the Dephosphorylated Form of BZR1.
(A) Western blot analysis of HY5 protein expression in the 35S::HY5-Myc/pBZR1::BZR1-CFP (Hy5-Myc/BZR1-CFP) transgenic plants. The asterisk denotes a non-specific band.
(B) Western blot analysis of HY5 protein expression in the 35S::HY5-Myc/pBZR1::mBZR1-CFP (Hy5-Myc/mBZR1-CFP) transgenic plants. mBZR1 is the BZR1 gene with the bzr1-1D mutation. The asterisk denotes a non-specific band.
(C and D) Transcript levels of BZR1 in the Hy5-Myc/BZR1-CFP (C) and Hy5-Myc/mBZR1-CFP (D) transgenic seedlings. Data are means ±SE. Pair-wise Student t tests were performed between different plants and the different letters (a, b, and c) above the bars indicate significance of the differences between them (a–b and b–c, P < 0.05; a–c, P < 0.01).
(E and F) Western blot assays of pBZR1 and BZR1 protein abundance in the Hy5-Myc/BZR1-CFP (E) and Hy5-Myc/mBZR1-CFP (F) transgenic plants grown under light (16-h light/8-h dark cycle at 22°C). In (A), (B), (E), and (F), Rubisco served as a loading control in the western blot analyses.
(G) BRZ responses of Hy5-Myc/mBZR1-CFP transgenic seedlings and control plants mBZR1-CFP and Col.
(H) Semi-in vivo pull-down assay of the HY5 interaction with pBZR1 and BZR1 in 35S::mBZR1-Myc transgenic seedlings treated with or without BL (10−6 M, 4 h). The BZR1-Myc proteins pulled down by GST-HY5 were detected by western blotting using an anti-Myc antibody.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
BR Deficiency Reduces the Transcription of Genes Involved in Cotyledon Development and Opening.
Transcript abundance of auxin (A), ethylene (B), and light (C) related genes that also control apical hook formation or cotyledon opening in the BR-deficient det2 mutant and its wild-type Col-0. UBC was used as an internal control for data normalization. Data are represented as means ± SD. Three biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: *P < 0.05; **P < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
HY5 Antagonizes BZR1 in Regulating the Transcription of Cotyledon Development and Opening Related Genes.
(A) A schematic map of the transient expression vector pGreenII-0800-LUC. REN, Renilla luciferase; LUC, firefly luciferase.
(B–E), Effects of HY5 on BZR1 transcriptional regulation of PIN1, LAX3, WAG2, and ACS5 promoters, respectively, in Col-0 protoplasts. In (B–E), data are means ±SD (n = 5 experiments). The asterisks indicate the levels of statistic significance determined by Student's t test: *P < 0.05; **P < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8
Transcript Levels of Four Cotyledon Development-Related Genes (PIN1, LAX3, WAG2, and ACS5) in Dark-Grown bzr1-1D and hy5-215 Mutants.
The 6-day-old dark-grown seedlings were collected for RNA extraction and expression analysis. UBC was used as an internal control for data normalization. Three biological repeats were performed. Data are shown as means ± SD. Pair-wise Student t tests were performed and the different letters (a–c) above the bars indicate significance of the differences between them (a–b and b–c, P < 0.01; a–c, P < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

10. Figure 9
A Model of BR and Light Signaling Crosstalk Mediated by BZR1 and HY5 Interaction during Photomorphogenesis.
(A) When seedlings grow in the light, HY5 is highly accumulated and the majority of BZR1 is present in the phosphorylated form and a small portion of dephosphorylated BZR1 is sequestered by HY5 and prevented from binding to its target genes for skotomorphogenic growth. As a result, the skotomorphogenic development of seedlings is suppressed and plants undergo photomorphogenic development with opened cotyledons and short hypocotyls.
(B) When seedlings grow in the dark, the accumulation of HY5 is remarkably diminished and most of the BZR1 proteins are in the dephosphorylated active form. Therefore, sequestration of the dephosphorylated BZR1 by HY5 is minimized and the active BZR1 can bind to its target gene promoters to promote skotomorphogenic processes such as elongation of hypocotyls and the formation of apical hooks with closed cotyledons.
(C) When seedlings grow under BR-deficient conditions in the dark, most of the BZR1 proteins are in the phosphorylated inactive form and the active form is captured by HY5, thus skotomorphogenic development of seedlings is repressed, which results in shortened hypocotyls and opened cotyledons.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions