Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors

Abstract

Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinase-mediated signal transduction pathway. Despite the identification of many components of this pathway, it remains unclear how the BR signal is transduced from the cell surface to the nucleus. Here we describe a complete BR signalling pathway by elucidating key missing steps. We show that phosphorylation of BSK1 (BR-signalling kinase 1) by the BR receptor kinase BRI1 (BR-insensitive 1) promotes BSK1 binding to the BSU1 (BRI1 suppressor 1) phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 (BR-insensitive 2) by dephosphorylating a conserved phospho-tyrosine residue (pTyr 200). Mutations that affect phosphorylation/dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR activation of BRI1, BSK1 and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR (brassinazole resistant) transcription factors in the nucleus. This study establishes a fully connected BR signalling pathway and provides new insights into the mechanism of GSK3 regulation.

Figure 1

BSU1 directly inhibits BIN2 phosphorylation of BZR1. (a) BR induces dephosphorylation of BIN2. Total proteins of TAP-BIN2 transgenic plants treated with 0.25 μM brassinolide (BL) or mock solution for 2 hrs were analyzed by two-dimensional gel electrophoresis followed by immunoblotting using the peroxidase anti-peroxidase antibody that detects TAP-BIN2. (b) BSU1 does not dephosphorylate phospho-BZR1 in vitro. BIN2-phosphorylated MBP-BZR1 (BIN2 removed) was incubated with GST, GST-BSU1 or GST-BSL1 for 12 hrs and analyzed by immunoblotting using anti-MBP antibody. (c–e) BSU1 inhibits BIN2 but not bin2-1. (c) GST-BIN2 was pre-incubated with GST-BSU1 or GST for indicated time before MBP-BZR1 and ³²P-γATP were added. (d) Partially phosphorylated ³²P-MBP-pBZR1 was further incubated with GST-BIN2, GST-BSU1, or both, in the presence of non-radioactive ATP, and analyzed by autoradiography. GST-BIN2 M115A is a kinase-inactive mutant BIN2. (e) GST-BIN2 or GST-bin2-1 was first treated with BSU1-YFP immunoprecipitated from BR-treated (+BL, 0.25 μM BL for 30 min) or untreated 35S::BSU1-YFP plants, and then incubated with MBP-BZR1 and ³²P-γATP. Col-0, immunoprecipitation from non-transgenic plant as control. CBB indicates Coomassie brilliant blue-stained gels. (f–i) BSU1 directly interacts with BIN2 and bin2-1. (f) Gel blot containing GST, GST-BIN2 and GST-bin2-1 was probed sequentially with MBP-BSU1 and anti-MBP antibody (upper) and then stained with Ponceau S (lower). (g) BiFC assay of interactions between BSU1 or BSL1 and BIN2. The indicated constructs were transformed into tobacco leaf cells. Bright spots in BIN2-nYFP+cYFP are chloroplast auto-fluorescence. Scale bar = 10 μm. (h) The proteins of tobacco leaves transiently transformed with the indicated constructs were immunoprecipitated with anti-GFP antibody, and the immunoblot was probed with anti-myc and anti-GFP antibody. (i) Arabidopsis plants (F1) expressing BSU1-YFP or co-expressing BSU1-YFP and BIN2-myc, grown on the medium containing BR biosynthetic inhibitor BRZ for 10 days, were treated with 10 μM MG-132 for 1 hr and then with 0.2 μM BL or mock solution for 15 min. Total protein extracts were immunoprecipitated with anti-myc antibodies, and the immunoblot was probed with anti-GFP and anti-myc antibodies. Full scan data of immunoblots and in vitro kinase/phosphatase assays are shown in Supplementary Information, Fig. S12.

Figure 2

BSU1 regulates BIN2 but not bin2-1 in vivo. (a) Subcellular localization of BZR1-YFP in the cells co-transformed with the indicated constructs. The scale bar is 10 μm. (b) Immunoblots of BZR1-YFP proteins obtained from the tobacco leaves co-transformed with constructs indicated. The upper band is phosphorylated BZR1 and lower one unphosphorylated. (c) Overexpression of BSU1-YFP reduces the accumulation of BIN2-myc protein in a transgenic Arabidopsis line. Heterozygous 35S::BIN2-myc and 35S::BIN2-myc/35S-BSU1-YFP plants (F1) were treated with 0.25 μM BL or mock solution for 30 min. Immunoblot was probed with anti-myc or anti-GFP antibodies, and a non-specific band serves as loading control. (d) BSU1 reduces the accumulation of BIN2 but not that of bin2-1. BIN2- or bin2-1-myc levels were analyzed by anti-myc antibody in tobacco cells co-expressing myc-tagged BSU1 or BSU1-D510N mutant protein. A nonspecific band serves as loading control. (e) Overexpression of BSU1-YFP (+BSU1) partially rescues the bri1-116 mutant, but not the bin2-1 mutant. (f) Hypocotyl phenotypes of seedlings (genotype shown) grown in the dark on MS medium for 5 days. Bottom two panels show confocal images of BSU1-YFP in the plants indicated. The scale bar is 10 μm. (g) Quantitative RT-PCR analysis of SAUR-AC1 RNA expression in wild type (bri1-116 (+/−)), bri1-116 (−/−), and BSU1-YFP/bri1-116 plants. Error bars indicate standard error. Full scan data of immunoblots are shown in Supplementary Information, Fig. S12.

Figure 3

BSU1 dephosphorylates pTyr200 of BIN2 but not bin2-1. (a) Tyr200 phosphorylation is required for BIN2 kinase activity. GST-BIN2 or GST-BIN2 Y200F was incubated with MBP-BZR1 and ³²P-γATP. CBB, Coomassie brilliant blue-staining. (b, c) BSU1 dephosphorylates pTyr200 of BIN2 but not of bin2-1. Immunoblots of GST-BIN2, GST-BIN2 Y200A and GST-bin2-1 mutant proteins, incubated with MBP or MBP-BSU1 (b) or with BSU1-YFP immunoprecipitated from transgenic Arabidopsis (c), were probed with the anti-pTyr antibody and then with anti-GST antibody. (%) indicates relative signal level of pTyr200 normalized to total GST-BIN2 or GST-bin2-1 protein. (d, e) BR induces dephosphorylation of pTyr200 of BIN2 but not bin2-1. (d) The det2 mutant was treated with 10 μM MG132 for 1 hr prior to treatment with 0.2 μM BL for the indicated time. BIN2 protein was immunoprecipitated with a polyclonal anti-serum and immunoblotted with anti-pTyr, anti-BIN2 serum, and anti-GSK3 α/β antibody. (e) Transgenic plants expressing BIN2-myc or bin2-1-myc was pretreated with 10 μM MG132 and then treated with 0.25 μM BL (+BL) or mock solution (-BL). BIN2-myc and bin2-1-myc were immunoprecipitated by anti-myc antibody and gel blots were probed with antibodies indicated. (f, g) Phosphorylation of Tyr200 is required for BIN2 inhibition of plant growth. (f) Overexpression of BIN2-YFP but not BIN2-Y200F-YFP causes severe dwarf phenotypes in T1 generation. Upper left panel shows zoom-in view. Lower panel shows BIN2-YFP and BIN2-Y200F protein levels detected by anti-GFP antibodies. A nonspecific band serves as loading control. (g) Overexpression of bin2-1-myc but not bin2-1-Y200F-myc caused dwarf phenotypes. Seventy-six of a total 281 35S::bin2-1-myc transgenic T1 seedlings and none of a total 412 35S::bin2-1-Y200F-myc transgenic plants showed dwarf phenotype. (h–j) BSU1 family members play an essential role in BR signaling. (h) Eight of 27 bsu1;bsl1 double mutant plants transformed with an artificial microRNA construct targeting BSL2 and BSL3 (BSL2,3-amiRNA) showed dwarf phenotypes. Right panel shows zoom-in view of the quadruple mutant. (i) Phenotypes of 5-day old dark-grown seedlings of bsu1;bsl1/BSL2,3-amiRNA. (j) Quantitative RT-PCR analysis of SAUR-AC1 RNA expression. Bars indicate standard error. Full scan data of immunoblots and in vitro kinase/phosphatase assays are shown in Supplementary Information, Fig. S12.

Figure 4

Regulation of the BIN2 homolog, AtSK12 by BSU1-mediated tyrosine dephosphorylation. (a) Phylogenetic tree of the ten Arabidopsis GSK3/Shaggy-like kinases (AtSKs). (b) Six AtSKs specifically interact with BZR1 in yeast two-hybrid assays. Activation domain (AD) fused AtSKs were transformed into the cells containing DNA binding domain (BD) fused BZR1. Yeast clones were grown on Synthetic Dropout (SD) or SD-Histidine medium. (c) Both AtSK12 and BIN2 interact with BZR1 in BiFC assays. Transgenic Arabidopsis plants expressing nYFP-BIN2, nYFP-AtSK12 and nYFP-AtSK12-cd (C-terminal 29 amino acid deletion) were crossed to BZR1-cYFP plants, respectively. The seedlings of F1 generation were grown in white light for 7 days and YFP signals of epidermal cells were observed. The scale bar is 10 μm. (d) Various phenotypes of transgenic plants (T1) overexpressing WT AtSK12 or AtSK12-E297K. (e) AtSK12 phosphorylates BZR1 in vitro. GST-AtSK12 was incubated with MBP-BZR1 and ³²P-γATP. CBB indicates Coomassie brilliant blue-stained gel. (f) BR induces degradation of AtSK12. Homozygous plants expressing AtSK12-myc were treated with 0.25 μM BL for 30 min. Proteins immunoprecipitated by anti-myc antibodies were blotted onto nitrocellulose membrane and probed by anti-myc antibody. (g) Overexpression of BSU1-YFP reduces the accumulation of AtSK12-myc protein in a transgenic Arabidopsis plant. (h) BR induces pTyr dephosphorylation of AtSK12. Homozygous AtSK12-myc plants were pretreated with 10 μM MG132 and then treated with 0.25 μM BL (+BL) or mock solution (-BL). AtSK12-myc was immunoprecipitated by anti-myc antibody and gel blots were probed with anti-pTyr and anti-myc antibodies. Full scan data of immunoblots and in vitro kinase/phosphatase assays are shown in Supplementary Information, Fig. S12.

Figure 5

BSK1 directly interacts with BSU1. (a) The GST fusion proteins of the kinase domains of BRI1 (GST-BRI1-K) and BAK1 (GST-BAK1-K) and full-length BSK1 (GST-BSK1) were separated by SDS-PAGE and blotted onto nitrocellulose membrane. The blot was probed sequentially with MBP-BSU1 and anti-MBP antibody (upper) and then stained with Ponceau S (lower). (b) BiFC assays show in vivo interaction between BSU1 or BSL1 and BSK1. Tobacco leaf epidermal cells were transformed with indicated constructs. At5g49760 is a receptor kinase unrelated to BR signaling used here as a negative control. Bright spots in nYFP+BSK1-cYFP and At5g49760-nYFP+BSK1-cYFP are chloroplast auto-fluorescence. The scale bar is 10 μm. (c) Total protein extracts obtained from Arabidopsis plants (F1) expressing BSU1-YFP or co-expressing BSU1-YFP and BSK1-myc were immunoprecipitated with anti-myc, and the immunoblot was probed with anti-GFP and anti-myc antibody. (d) Phosphorylation of BSK1 Ser230 by BRI1 enhances BSK1 binding to BSU1. GST-BSK1 or GST-BSK1 S230A was incubated with GST-BRI1-K or GST for 2 hrs. Overlay assay was performed as described in (a). Full scan data of immunoblots are shown in Supplementary Information, Fig. S12. (e) The BR signal transduction pathway. Filled objects indicate components in active states and open objects inactive states. In the absence of BR (-BR), BRI1 is kept in an inactive form with help of its inhibitor BKI1, and consequently BAK1, BSK1 and BSU1 are inactive, while BIN2 is active and phosphorylates BZR1 and BZR2 (BZR1/2), leading to their loss of DNA binding activity, exclusion from the nucleus by the 14-3-3 proteins, and degradation by the proteasome. In the presence of BR (+BR), BR binding to the extracellular domain of BRI1 induces dissociation of BKI1 and association and inter-activation between BRI1 and BAK1. Activated BRI1 then phosphorylates BSK1, which in turn dissociates from the receptor complex and interacts with and presumably activates BSU1. BSU1 inactivates BIN2 by dephosphorylating its pTyr200, allowing accumulation of unphosphorylated BZR1/2, likely with help of an unknown phosphatase. Unphosphorylated BZR1/2 accumulate in the nucleus and bind to promoters to regulate the expression of BR-target genes, leading to cellular and developmental responses.