Volume 10, Issue 2, Pages 297-308 (February 2017) BKI1 Regulates Plant Architecture through Coordinated Inhibition of the Brassinosteroid and ERECTA Signaling Pathways in Arabidopsis  Dongxu Wang, Cangjing Yang, Haijiao Wang, Zhihua Wu, Jianjun Jiang, Jingjing Liu, Zhuona He, Fang Chang, Hong Ma, Xuelu Wang  Molecular Plant  Volume 10, Issue 2, Pages 297-308 (February 2017) DOI: 10.1016/j.molp.2016.11.014 Copyright © 2017 The Author Terms and Conditions

Figure 1 BKI1 Interacts with ER Family Proteins. (A) BKI1-MBP interacted with ER-KC-GST and ERL1-KC-GST in the GST pull-down assays. The Coomassie brilliant blue (CBB) staining shows equal loading. Asterisk indicates unspecific bands. (B) BKI1-MBP, BKI1-N-MBP, and BKI1-C-MBP interact with ER-KC-HIS in the MBP pull-down assays. mBAK1-CD-HIS was used as a negative control. (C) The interaction of BKI1 with ER-KC-GST and ERL1-KC-GST detected by semi-in vivo pull-down assays. Protein extracts from the BKI1-FLAG plants were used. (D) The interaction of BKI1-YFP with ER-FL-FLAG in vivo. Total proteins were extracted from plants overexpressing both ER-FL-FLAG and BKI1-YFP. The ER-FL-FLAG was immunoprecipitated with anti-FLAG agarose gel and the coIPed BKI1-YFP was detected by immunoblotting with anti-GFP antibody. (E and F) GUS staining of the 14-day-old seedlings (E) and the 6-week-old inflorescences (F) of both ProBKI1:GUS and ProER:GUS lines. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions

Figure 2 The Loss of Function of ER Strongly Enhances the Dwarf Phenotype of the BKI1-YFP Overexpression Line. (A) Rosette phenotypes of the Col-0, er-105, BKI1-YFP, BKI1-YFP er-105, bri1-301, and bri1-301 er-105 plants. (B) Quantification of the rosette radius in (A) (n = 25). (C) Mature plant phenotypes of the Col-0, er-105, BKI1-YFP, BKI1-YFP er-105, bri1-301, and bri1-301 er-105 plants. (D) Quantification of the plant height in (C) (n = 25). In (B) and (D), error bars indicate SEM. Student's t-tests were used to determine significant levels of the indicated comparisons.***p < 0.001; NS, no significance; n, number of plants analyzed. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions

Figure 3 BKI1 Acts with the ER Signaling Pathway to Regulate Pedicel Orientation. (A) Inflorescence architecture of the 6-week-old Pro35S:BKI1-YFP lines and Col-0. (B) Expression levels of BKI1 in (A). Total RNA was extracted from the 10-day-old seedlings. (C) Quantification of the pedicel orientation in (A) (n = 15). (D) Inflorescence architecture of the 6-week-old Col-0 and bki1-1 plants. (E) Quantification of the pedicel orientation in (D) (n = 15). (F) Inflorescence architecture of the 6-week-old Col-0, er-105, er-105 erl2-1, er-105 erl2-1 erl1-2(+/−), er-105 erl2-1 cll1 cll2, er-105 erl2-1 cll1 cll2 chal (+/−), er-105 erl2-1 cll1 cll2 chal, and ER-Δkinase-FLAG/er-105 plants. (G) Quantification of the pedicel orientation in (F) (n = 15). (H) Inflorescence architecture of the 6-week-old Col-0, er-105, BKI1-YFP, and BKI1-YFP er-105 plants. (I) Quantification of the pedicel orientation in (H) (n = 15). (J) Inflorescence architecture of the 6-week-old Col-0, bki1-1, er-105 erl2-1, and bki1-1 er-105 erl2-1 plants. (K) Quantification of the pedicel orientation in (J). (L) The seedling and inflorescence architecture of plants overexpressing the ProER-Ω:MKK5DD-FLAG in the BKI1-YFP line. Three independent transgenic lines were used. The lower panels show the corresponding protein levels detected by western blotting with FLAG and GFP antibodies. The Ponceau-S stained Rubisco large subunit was used as a loading control. In (B), (C), (E), (G), (I), and (K), error bars indicate SE. Student's t-tests were used to determine significant levels of the indicated comparisons. ***p < 0.001, *p < 0.05; NS, no significance. n, number of plants analyzed. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions

Figure 4 Most of the Common Differentially Expressed Genes in BKI1-YFP and er-105 Show Similar Changes in Expression. (A and B) Venn diagrams show the number of differentially expressed genes (A) between the er-105 mutant and the BKI1-YFP line, or (B) between the bri1-301 mutant and the BKI1-YFP line. These genes are illustrated in two subgroups: genes regulated in the same (co) or opposite direction (anti). (C) Heatmap showing subgroup genes regulated in both the er-105 mutant and the BKI1-YFP line. (D) Heatmap showing subgroup genes regulated in both the bri1-301 mutant and the BKI1-YFP line. In (C) and (D), co-up denotes genes upregulated in both backgrounds; co-down denotes genes downregulated in both backgrounds; anti denotes genes regulated in reverse directions between two backgrounds. (E) Log2 value of the BKI1 expression fold changes in the er-105 and bri1-301 mutants compared with Col-0. Error bars indicate SE. (F) Correlation between the expression levels of 30 randomly selected genes and BKI1 determined by RNA-seq and qRT–PCR. The x axis gives the log2 value of the fold change detected by qRT–PCR and the y axis gives the log2 value detected by RNA-seq. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions

Figure 5 BKI1 Acts as a Negative Regulator to Modulate ER Signaling. (A) Effect of BKI1 on the autophosphorylation of ER-KC and the phosphorylation of the ER substrate mBAK1-CD (kinase-dead mutant) analyzed by a general anti-pThr antibody. The CBB staining and signals detected by HIS antibody show equal loading. Asterisk indicates unspecific bands. (B) Quantification of the relative phosphorylation level of ER-KC-HIS and mBAK1-CD-HIS in (A) (n = 3). The phosphorylation levels of ER-KC-HIS in line 1 and mBAK1-CD-HIS in line 3 were defined as “1,” respectively. (C) The phosphorylation assays of BKI1 by ER and ERL1 kinases. BKI1 phosphorylation by BRI1-JKC-HIS was used as a positive control. Proteins were incubated with [γ-32P]ATP and separated by SDS–PAGE followed by autoradiography. (D) Subcellular localization of BKI1-YFP. The 4-day-old seedlings expressing the estrogen-inducible EPF1-FLAG (Est:EPF1-FLAG) or EPF2-FLAG (Est:EPF2-FLAG) in BKI1-YFP were treated with 10 μM β-estradiol or DMSO. Scale bar, 10 μm. (E) The interaction of BKI1-YFP with ER-FL-FLAG under eBL treatment. Total proteins were extracted from plants overexpressing both ER-FL-FLAG and BKI1-YFP after treatment with 5 μM eBL (+) or DMSO (−) for 1 h. The ER-FL-FLAG was immunoprecipitated with anti-FLAG agarose gel and the coIPed BKI1-YFP was detected by immunoblotting with anti-GFP antibody. (F) MPK activities in the BKI1-YFP and BKI1S270/274A-GFP transgenic plants. The 10-old-day seedlings were treated with 5 μM eBL (+) or DMSO (−) for 1 h. Immunoblots were conducted with an anti-p44/42-ERK antibody (Cell Signaling Technology). MPK6, MPK3, and MPK4 were identified by their molecular mass and are indicated by arrows. The Ponceau-S stained Rubisco large subunit was used as a loading control. The relative phosphorylation levels of MPKs were quantified (n = 3). The pMPK levels in BKI1-YFP under DMSO treatment in line 1 were defined as “1.” In (B) and (F), error bars indicate SE. Student's t-tests were used to determine significant levels of the indicated comparisons. **p < 0.01, *p < 0.05; NS, no significance. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions

Figure 6 A Proposed Model Depicting the Role of BKI1 as a Suppressor of Both ER and BRI1 Signaling Pathways. (A) In the absence of BRs, BKI1 suppresses the BRI1 and ER receptor kinases. In the BR signaling pathway, BKI1 inhibits BRI1 and blocks downstream signaling to affect plant architecture. In the ER signaling pathway, BKI1 also inhibits ER activity and its downstream MAPK cascade to regulate pedicel orientation and other aspects of plant architecture. ER signaling did not phosphorylate BKI1 or induce its dissociation from the plasma membrane. (B) In the presence of BRs, the activated BRI1 phosphorylates BKI1 to promote its dissociation from the plasma membrane, leading to a loss of inhibition on both BRI1 and ER. ER signaling was enhanced (the thickened arrows represent the enhanced ER signaling). In addition, both ER and BR signaling induced BKI1 transcription by a negative feedback loop. “P” represents the phosphorylation state. The black arrows represent direct regulation at the protein level. The dotted black and gray arrows represent indirect regulation at the protein and transcription level, respectively. Molecular Plant 2017 10, 297-308DOI: (10.1016/j.molp.2016.11.014) Copyright © 2017 The Author Terms and Conditions