Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase

Abstract

Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development.

Keywords: BIN2; HDA6; brassinosteroid signaling; deacetylation; development.

Fig. 1.

HDA6 interacts with and deacetylates BIN2. (A) HDA6 interacts with BIN2 in BiFC assays. The nYFP-BIN2 or nYFP and HDA6-cYFP or cYFP constructs were cotransformed into the pavement cells of N. benthamiana. BF, bright field; magnification, 20×. (B) HDA6-GST interacts with BIN2-His in GST-pull down assays. (C) HDA6-YFP coimmunoprecipitated by BIN2-FLAG in plants. The asterisk indicates a nonspecific band. (D) The recombinant BIN2-His protein purified from E. coli was acetylated. The E. coli strain containing the BIN2-His construct was cultured in LB medium with or without 5 nM TSA and 5 mM NAM. The acetylation level of the purified BIN2-His was detected with an anti–acetyl-Lys antibody. (E) BIN2-His can be deacetylated by HDA6 in vitro. The acetylation status of BIN2-His was determined with an anti–acetyl-Lys antibody. (F) Statistical analysis of the relative acetylation level of BIN2-His. (G) BIN2-FLAG is acetylated in plants. BIN2-FLAG and HDA6-YFP BIN2-FLAG (crossed from BIN2-FLAG and HDA6-YFP) plants were grown on soil for 2 wk. The acetylation level of the immunoprecipitated BIN2-FLAG protein was detected with an antiacetyl-Lys antibody. Error bars represent SE.

Fig. S1.

HDA6 regulates BR signaling through the BIN2 kinase. (A) BIN2 cannot phosphorylate HDA6. (B) Hypocotyl lengths of dark-grown bri1-301 (n = 25), axe1-5 bri1-301 (n = 25), and HDA6-YFP bri1-301 (n = 25) on medium containing 1 μM BRZ220. (C) HDA6 can deacetylate bin2-1 protein. Error bars represent SE. ***P < 0.001.

Fig. 2.

HDA6 positively regulates BR signaling. (A) Dark-grown hypocotyl phenotypes of Col-0, the axe1-5 mutant, and the HDA6-YFP overexpression line. (B) Hypocotyl lengths of the dark-grown Col-0 (n = 25), axe1-5 (n = 25), and HDA6-YFP (n = 25). (C) Expression levels of the BR-responsive genes CPD and DWF4. Their expression level in Col-0 was defined as “1.” (D) The seedling phenotype of Col-0, BRI1-GFP, and HDA6-YFP plants grown on medium containing 1 μM BR synthetic inhibitor BRZ220 and propiconazole (PCZ). (E) Phosphorylation status of BES1 in Col-0, BRI1-GFP, and HDA6-YFP plants. BES1 was detected by an anti-BES1 antibody. Error bars represent SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.

HDA6 regulates BR signaling through BIN2 and its homologs. (A–C) Hypocotyl length of Col-0 and the BRI1-GFP line (A), Ws-2 and bin2-3 bil1 bil2 plants (B), and En-2 and bes1-D plants (C). Plants were grown in the dark on medium containing different concentrations of TSA. (D) HDA6 partially rescued the dwarf phenotype of bri1-301. (E) HDA6 can partially suppress the dwarf phenotype of bin2-1. (F) Expression levels of the BR-responsive genes CPD and DWF4 in bri1-301, axe1-5 bri1-301, and HDA6-YFP bri1-301. Their expression in bri1-301 was defined as 1. (G) Expression levels of the BR-responsive genes CPD and DWF4 in bin2-1, axe1-5 bin 2-1, and HDA6-YFP bin 2-1. Their expression in bin-2-1 was defined as 1. (H) HDA6-RNAi cannot suppress the seedling phenotype of the bin2-3 bil1 bil2 triple mutant. (I) Expression levels of CPD, DWF4, and HDA6 in bin2-3 bil1 bil2 and HDA6-RNAi bin2-3 bil1 bil2 plants. Their expression level in Ws-2 was defined as 1. (J) HDA6-RNAi hardly affects hypocotyl elongation in the bin2-3 bil1 bil2 background growing on medium containing 1 μM BRZ220 in the dark. Error bars represent SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.

HDA6 deacetylates BIN2 to inhibit its kinase activity. (A) Acetylated BIN2 has higher kinase activity to phosphorylate BES1-MBP. (B) Acetylation levels of the mutated BIN2 proteins on the predicted acetylation residues. BIN2^K189R showed reduced acetylation level and autophosphorylation activity compared with other forms of BIN2. BIN2^K69R showed unphosphorylated BIN2. (C) Additional HDA6 did not change the acetylation level of BIN2^K189R. (This acetylation band of BIN2^K189R may be caused by nonspecific recognition of the antibody or acetylation of BIN2 by other enzymes on other residues.) (D) BES1-MBP phosphorylation by BIN2, BIN2^K5R, BIN2^K189R, and BIN2^K347R. (E) Acetylation level of BIN2-FLAG and BIN2^K189R-FLAG from plants with or without TSA treatments. (F) Statistical analysis of the relative acetylation level of BIN2-FLAG and BIN2^K189R-FLAG from plants with or without TSA treatments. The acetylation level of BIN2-FLAG was defined as 1. (G) bin2-1^K189R-FLAG overexpression rescued the dwarf phenotype of bin2-1-FLAG overexpression. (H) Expression levels of the BR-responsive genes CPD and DWF4 in BIN2-FLAG, BIN2^K189R-FLAG, bin2-1-FLAG, and bin2-1^K189R-FLAG plants. (I) BES1 phosphorylation status in BIN2-FLAG, BIN2^K189R-FLAG, bin2-1-FLAG, and bin2-1^K189R-FLAG plants detected by an anti-BES1 antibody. (J) Acetylation levels of BIN2-His, BIN2^K189R-His, bin2-1-His, and bin2-1^K189R-His proteins purified from E. coli. (K) Phosphorylation of BES1-MBP by BIN2-His, BIN2^K189R-His, bin2-1-His, and bin2-1^K189R-His protein. Error bars represent SE. *P < 0.05 and ***P < 0.001.

Fig. S2.

Identification of the acetylation sites of BIN2 by LC-MS/MS. (A) K5 is a potential acetylation site of BIN2. (B) K189 is a potential acetylation site of BIN2. (C) K347 is a potential acetylation site of BIN2.

Fig. S3.

Energy affects the acetylation of BIN2. (A) Seedling phenotype of Ws-2 and bin2-3 bil1 bil2 grown in the dark on medium with or without TSA and glucose or sucrose. (B) Glucose enhances BIN2-His acetylation. (C) ATP inhibits the interaction between HDA6 and BIN2.

Fig. S4.

Proposed model. BIN2, an important negative regulator in the BR signaling pathway, is regulated by upstream BR signals through phosphorylation. Phosphorylated and acetylated BIN2 has higher activity to phosphorylate the downstream transcription factors BES1 and BZR1 to inhibit BR signaling. Under energy-limited conditions, HDA6 interacts with BIN2 and deacetylates BIN2 on the K189 site to inhibit BIN2 activity and promote BR signaling. The expression of HDA6 can be feedback-inhibited, likely through BES1- and BZR1-mediated transcription regulation. HDA6 may also interact with BES1 to regulate its transcription activity.

Fig. S5.

HDA6 interacts with BES1. (A) HDA6-GST interacts with BES1-MBP in GST-pull down assays. (B) BES1 interacts with HDA6-FLAG in plants.