Volume 11, Issue 2, Pages 315-325 (February 2018) Abscisic Acid Signaling Inhibits Brassinosteroid Signaling through Dampening the Dephosphorylation of BIN2 by ABI1 and ABI2  Haijiao Wang, Jie Tang, Jing Liu, Jin Hu, Jingjing Liu, Yuxiao Chen, Zhenying Cai, Xuelu Wang  Molecular Plant  Volume 11, Issue 2, Pages 315-325 (February 2018) DOI: 10.1016/j.molp.2017.12.013 Copyright © 2017 The Author Terms and Conditions

Figure 1 Expression of ABI2 Enhances BR Signaling Outputs. (A and B) Phenotypes of the plants expressing the pABI2::ABI2-FLAG and pABI2::abi2-1-FLAG in Col-0 (A) and bri1-301 (B). The seedlings were grown under light for 3 weeks. The protein levels in the transgenic plants were detected by western blotting with anti-FLAG antibody. The Ponceau-S-stained Rubisco large subunit served as the loading control. (C and D) The hypocotyl phenotypes (C) and length (D) of the plants expressing pABI2::ABI2-FLAG in Col-0 and bri1-301 backgrounds. The seedlings were grown in the dark for 4 days. (E and F) The expression levels of the BR-inhibited gene DWF4 and the BR-induced gene SAUR-AC1 in the plants carrying pABI2::ABI2-FLAG in the Col-0 (E) or bri1-301 (F) background. Error bars indicate SE in (D) and SD in (E) and (F). The significance of differences was measured by Student's t-test. ***p < 0.001; **p < 0.01; *p < 0.05. (G and H) The BES1 phosphorylation status in the plants carrying pABI2::ABI2-FLAG in the Col-0 (G) or bri1-301 (H) background. (I) BES1 phosphorylation status in pABI2::abi2-1-FLAG and abi2-3 mutant plants. BES1 was detected by anti-BES1 antibody. In (G) and (I), the Rubisco large subunit was used as loading control. In (H), the loading was measured by anti-tubulin antibody. The numbers indicate the ratio of the unphosphorylated BES1 to the phosphorylated BES1. (J) Differentially expressed genes between abi2-3 mutant and Col-0 with 1.0 μM eBL treatment for 1 h. (K) Histograms show percentage of the BR-regulated genes in the abi2-3 mutant. The x axis shows the log2 expression levels of genes in abi2-3 mutant. Numbers in boxes indicate percentage sum of upregulated (orange) and downregulated (green) genes. BR_UP and BR_DOWN indicate the BR-upregulated and -downregulated genes after eBL treatment, respectively. See also Supplemental Figure 1. Molecular Plant 2018 11, 315-325DOI: (10.1016/j.molp.2017.12.013) Copyright © 2017 The Author Terms and Conditions

Figure 2 ABI1 and ABI2 Interact with BIN2. (A) The interaction between ABI1, ABI2, and BIN2 was tested by BiFC assays. The indicated constructs were transformed into the leaves of Nicotiana benthamiana. Scale bars, 50 μm. (B) The interaction of ABI1 and ABI2 with BIN2 was detected by in vitro pull-down assays. The GST-ABI1/ABI2 and His-BIN2 fusion proteins were expressed and purified from Escherichia coli. Anti-His antibody was used to detect the His-BIN2 protein. Anti-GST antibody was used to detect the amount of GST-ABI1/ABI2 protein. (C) The interaction of ABI1 and ABI2 with BIN2 was detected by semi-in vivo pull-down assays. The GST-ABI1/ABI2 fusion protein was expressed and purified from E. coli. Protein extracts from the plants overexpressing BIN2-FLAG were used for the pull-down assays. The loading of the related recombinant proteins was shown by Coomassie brilliant blue (CBB) staining. See also Supplemental Figure 2. Molecular Plant 2018 11, 315-325DOI: (10.1016/j.molp.2017.12.013) Copyright © 2017 The Author Terms and Conditions

Figure 3 ABI1 and ABI2 Dephosphorylate BIN2 to Inhibit BES1 Phosphorylation. (A) Dephosphorylation of BIN2 by ABI2 was detected by immunoblotting. Anti-His antibody was used to detect the phosphorylation status of the His-BIN2 and His-BIN2K69R proteins; the GST-ABI2 protein was detected by anti-GST antibody. (B) ABI2 dephosphorylated BIN2 detected by anti-pSer antibody. The lower blot shows the loading by Coomassie brilliant blue (CBB) staining. (C) Dephosphorylation of BIN2 by ABI1, abi1-1, ABI2, and abi2-1 was detected by [γ-32P]ATP. The upper blot shows autoradiography. The lower blot shows the CBB staining to indicate the loading of recombinant proteins. The numbers are the quantitative analysis of the signal intensity. The abundance of GST treatment was set as 1 for calculating the relative abundance. (D) ABI1 and ABI2 inhibited phosphorylation of BES1 by dephosphorylating BIN2 kinase. The phosphorylation status of BES1 was detected by anti-MBP antibody. The amounts of recombinant proteins His-BIN2 and GST-ABI2 were determined by anti-His antibody and CBB, respectively. (E) ABI2 cannot dephosphorylate phosphorylated BES1 (pBES1). The phosphorylation status of BES1 was detected by anti-MBP antibody. Anti-GST antibody was used to detect the GST protein loading. See also Supplemental Figures 3 and 4. Molecular Plant 2018 11, 315-325DOI: (10.1016/j.molp.2017.12.013) Copyright © 2017 The Author Terms and Conditions

Figure 4 ABA Regulates the Dephosphorylation of BIN2 by Its Primary Signaling. (A) Reconstruction of the ABA signaling pathway to determine BIN2 dephosphorylation by ABI2 and abi2-1 in vitro. The protein levels were examined by western blotting with the corresponding antibodies. (B) ABA treatment can enhance the accumulation of the phosphorylated BES1. Calf Intestinal Alkaline Phosphatase (CIP) treatment was used to dephosphorylate BES1. The phosphorylation status of BES1 was detected by anti-MBP antibody. The BIN2-FLAG protein was detected by anti-FLAG antibody. * indicates the IgG heavy chain. (C and D) The hypocotyl phenotypes (C) and length (D) of the pyr4- mutant in the Col-0 and bri1-301 backgrounds. The seedlings were grown in the dark for 4 days. (E) The expression levels of the BR-suppressed genes CPD and DWF4. Error bars indicate SE in (D) and SD in (E). The significance of differences was measured by Student's t-test. ***p < 0.001; **p < 0.01; *p < 0.05. (F) Differentially expressed genes between ABA and eBL treatment in Col-0(er) and Col-0, respectively. The 9-day-old seedlings were treated either by 10 μM (±)-ABA dissolved in ethanol or by eBL dissolved in DMSO for 1 h. (G) Histograms showed percentage of the BR-regulated genes in Col-0. The x axis shows the log2 expression levels of co-regulated genes by ABA and eBL after ABA treatment. Numbers in boxes indicate percentage sum of upregulated (orange) and downregulated (green) genes. BR_UP and BR_DOWN indicate BR-upregulated and -downregulated genes after eBL treatment, respectively. (H) Differentially expressed genes between ABA and eBL treatment in pyr4- or Col-0, respectively. Molecular Plant 2018 11, 315-325DOI: (10.1016/j.molp.2017.12.013) Copyright © 2017 The Author Terms and Conditions

Figure 5 GSK3s Are Involved in ABA Responses. (A) Differentially expressed genes between bikinin and ABA long-term treatment. For bikinin treatment, the 9-day-old seedlings were collected from 5 μM bikinin-containing plates. For the long-term ABA treatment, 9-day-old seedlings were collected from plates supplemented with 100 nM ABA. (B) Histograms show percentage of the long-term ABA-regulated genes in bikinin treatment. The x axis shows the log2 expression levels of genes co-regulated by bikinin and ABA after long-term ABA treatment in Col-0. Numbers in boxes indicate percentage sum of upregulated (orange) and downregulated (green) genes. ABA long_UP and ABA long_DOWN indicate ABA upregulated genes and ABA downregulated genes after long-term ABA treatment. (C) GO term analysis of the biological processes enriched in ABA long-term upregulated genes and bikinin downregulated genes. (D) A proposed model to illustrate how ABA early signaling enhances GSK3s activity by inhibiting ABI1 and ABI2 to both suppress BR signaling and promote ABA signaling. Without ABA, ABI1 and ABI2 inhibit the activity of GSK3 by dephosphorylation, and BR signaling remains at a higher level; meanwhile, GSK3 cannot activate SnRK2s to keep ABA signaling outputs at a lower level, resulting in normal plant growth and development. With ABA, the ABA receptors will bind ABI1 and ABI2 to remove their inhibition on the kinase activity of GSK3, which leads to a reduction of BR signaling outputs to inhibit plant growth and enhanced SnRK2s activity and ABA signaling outputs, to promote stress responses and adapt to unfavorable growth conditions. Molecular Plant 2018 11, 315-325DOI: (10.1016/j.molp.2017.12.013) Copyright © 2017 The Author Terms and Conditions