1. Volume 21, Issue 5, Pages 825-834 (November 2011)
Dual Role of BKI1 and  s in Brassinosteroid Signaling to Link Receptor with Transcription Factors 
Haijiao Wang, Cangjin Yang, Chi Zhang, Niyan Wang, Dihong Lu, Jie Wang, Shanshan Zhang, Zhi-Xin Wang, Hong Ma, Xuelu Wang 
Developmental Cell 
Volume 21, Issue 5, Pages (November 2011)
DOI: /j.devcel
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2. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
The Amino and Carboxyl Regions of BKI1 Have Distinct Functions
(A and B) The phenotype of plants overexpressing the empty vector (V), the full-length BKI1-YFP (FL), the BKI1-N-YFP (N), and the BKI1-C-YFP (C) in the Col-0 (A) and bri1-301 (B) backgrounds. The lower panels show the corresponding protein levels. Ponceau-S Stained Rubisco large subunit serves as a loading control.
(C–F) The expression levels of some BR-responsive genes in various BKI1-related transgenic plants, Col-0, and bri CPD (C), DWF4 (D), and Saur-AC1 (E) are in the Col-0 background. The seedlings were treated with 1.0 μM eBL for 1 hr. (F) The expression levels of CPD, DWF4, and Saur-AC1 in the bri1-301 background. All error bars indicate standard deviation (SD). Asterisks indicate a statistically significant difference from the wild-type (p < 0.05); pounds indicate a significant difference from the eBL treated control (p < 0.05).
See also Figure S1 and Table S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Cytosolical Localization of the Carboxyl Termini of BKI1 Enhances BR Signaling
(A–I) The subcellular localization of BKI1-YFP (A–C), BKI1-N-YFP (D–F), and BKI1-C-YFP (G–I) before and after eBL treatment in Col-0 background. Scale bars, 20 μm. Seedlings were grown on 1/2 Murashige & Skoog (MS) medium for 4 days. (A), (D), and (G) were untreated; (B), (E), and (H) were treated with 1 μM eBL for 5 min; and (C), (F), and (I) were treated with 1 μM eBL for 10 min. See also Figure S2.
(J) The phenotype of plants expressing BKI1-C fused to a nuclear localization signal (NLS) or a nonfunctional NLS (nls) in bri1-301 background. The T1 seedlings were grown under dim light for 14 days.
(K) The accumulation of BES1 in the pooled larger plants and the pooled bri1-301-like plants was detected by western blotting with anti-BES1 antibody. Ponceau-S Stained Rubisco large subunit serves as a loading control.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
The S270 and S274 Sites of BKI1 Play Essential Roles in BKI1 Dissociation from the Plasma Membrane
(A) The phosphorylation status of BKI1-FLAG on Serine residues in seedlings with or without eBL treatment. Immunoprecipitation (IP) with anti-FLAG agarose; immunoblotting with anti-pSer or anti-FLAG antibody. The seedlings were grown on 1/2 MS medium containing 1 μM BRZ220 and were treated with 5 μM eBL or mock (DMSO) for 1 hr.
(B) The subcellular localization of BKI1S270/274A-GFP, BKI1S270A-YFP, and BKI1S274A-YFP proteins in Col-0 before and after 1 μM eBL treatment. The time of each treatment is indicated. Scale bars, 10 μm.
(C) The phenotype of plants overexpressing BKI1S270/274A-GFP, BKI1S270A-YFP or BKI1S274A-YFP in Col-0 background. Their protein levels were detected by western blotting with anti-GFP antibody.
(D) The BES1 accumulation status in the BKI1-YFP and BKI1S270/274A-GFP transgenic plants. Immunoblotting with anti-BES1 antibody shows levels of the phosphorylated BES1 (p-BES1) and the dephosphorylated BES1. The 14-old-day seedlings were treated with 1 μM eBL (+) or DMSO (−) for 1 hr. In (C) and (D), Ponceau-S Stained Rubisco large subunit serves as a loading control.
(E) Phosphorylation assay of wild-type and mutated BKI1 by BRI1 kinase. An equal amount of recombinant proteins, as indicated by the Coomassie Brilliant Blue (CBB) staining (bottom), was incubated with [γ-32P] ATP and separated by SDS-PAGE, followed by autoradiography (top).
See also Figure S3 and Table S2.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
eBL Treatment Enhances the Interaction of BKI1 with κ In Vitro and In Vivo
(A) The interaction of BKI1 with λ and κ was detected by semi-in vivo pull-down assay λ and κ fused with GST were expressed and purified from E. coli. Protein extracts from plants overexpressing BKI1-FLAG was used for pull-down assay.
(B) The interaction between BKI1 and κ was tested by BiFC assay. The indicated constructs were transformed into leaves of Nicotiana benthamiana. Scale bars, 20 μm.
(C) The interaction of BKI1-YFP with κ-FLAG was detected by Co-IP. The κ-FLAG was IPed with anti-FLAG agarose, and the Co-IPed BKI1-YFP was detected by immunoblotting with anti-GFP antibody.
(D) Two known binding sequences (mode I and mode II) are aligned against the BKI1 sequence. “X” presents any given amino acid. The phosphoserine residues are marked with bold font.
(E) The interaction of BKI1S270A/274A with κ was detected with a semi-in vivo pull-down assay.
(F) The in vivo interaction of BKI1S270A/274A with κ was detected by Co-IP.
(G) The interaction of BKI1S270A and BKI1S274A with κ was detected by semi-in vivo pull-down assay.
In (A), (C), and (F), the 14-day-old seedlings were treated with 5.0 μM eBL (+) or DMSO (−) for 1 hr. An equal amount of recombinant proteins were indicated by the Coomassie brilliant blue (CBB) staining (bottom) in the semi-in vivo pull-down assay. See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
Overexpression of κ Can Release the Inhibitory Effect of BKI1 on Plant Growth
(A) The interaction of BKI1-C-YFP with κ-FLAG was detected by Co-IP.
(B) The phosphorylation assay of the truncated BKI1s by BRI1 kinase. An equal amount of recombinant protein, as indicated by the Coomassie brilliant blue (CBB) staining (bottom), was incubated with [γ-32P] ATP and separated by SDS-PAGE, followed by autoradiography (top).
(C) The phenotype of the κ-FLAG overexpression lines in Col-0 background. The protein levels of κ-FLAG were detected by western blotting with anti-FLAG antibody.
(D–F) The phenotype of plants overexpressing κ-FLAG with BKI1-YFP (D), BKI1S270/274A-GFP (E), or BKI1S270A-YFP (F). The expression levels of transgenes were detected by western blotting with anti-FLAG and anti-GFP antibody.
(G) The subcellular localization of BKI1-YFP or BKI1S270A-YFP in the pavement cells of Nicotiana benthamiana.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6 The Phosphorylated BKI1 Competes 14-3-3κ Away from BES1
(A) The in vivo interaction of κ-FLAG with BES1. The protein extracts from the κ-FLAG line were IPed with anti-FLAG agarose, and the resulting precipitates were detected by western blotting with anti-FLAG or anti-BES1 antibody. The 14-day-old seedlings were treated with DMSO (−) or 5.0 μM eBL (+) for 1 hr.
(B) The level and phosphorylation status of BES1 in cytosolic (C) or nuclear (N) fractions from the BKI1-C overexpression lines. The western blotting with anti-BES1 antibody was used to detect BES1. Histone H2B was used as a nuclear marker.
(C) The hypocotyl length of dark-grown seedlings on 1/2 MS medium containing different concentrations of BRZ220 for 4 days. The “FL-High” line is the commonly used BKI1-YFP overexpression line in our study. See also Figure S6.
(D) The effect of phosphorylated BKI1 on the interaction of κ with BES1. The protein extracts from the κ-FLAG plants were IPed with anti-FLAG agarose. MBP fused BKI1 and BKI1S270/274A proteins (5 μg) were preincubated with BRI1 kinase (0.5 μg) with or without 10 mM ATP for 30 min, and then they were added to the IPed κ-FLAG products and incubated for 2 hr. The proteins bound with anti-FLAG agarose were eluted and detected by western blotting with anti-BES1 antibody. The inputs of MBP fused proteins and κ were detected by western blotting with anti-MBP and anti-FLAG antibody, respectively. The input of BES1 was determined by western blotting with anti-BES1 antibody.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7 A Model of the BR Signal Transduction Pathway
(A) In the absence of BRs, BKI1 proximal to the plasma membrane keeps BRI1 in an inactive state through their direct interaction; BIN2 phosphorylates BES1 and BZR1 and inhibits their DNA binding activity; proteins keep the phosphorylated BES1/BZR1 in the cytoplasm.
(B) BR binding to BRI1 activates BRI1 kinase, which promotes BKI1 phosphorylation. The phosphorylated BKI1 dissociates from plasma membrane and BRI1, and then binds to proteins in cytosol to free BES1/BZR1. The released BES1/BZR1 accumulates in the nucleus, leading to BR-responsive gene expression. Stars represent the phosphorylation state.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions