Sumoylation of BRI1-EMS-SUPPRESSOR 1 (BES1) by the SUMO E3 Ligase SIZ1 Negatively Regulates Brassinosteroids Signaling in Arabidopsis thaliana

Abstract

Brassinosteroids (BRs), a group of plant steroid hormones, participate in the regulation of plant growth and developmental processes. BR functions through the BES1/BZR1 family of transcription factors, however, the regulation of the BES1 activity by post-translational modifications remains largely unknown. Here, we present evidence that the SUMO E3 ligase SIZ1 negatively regulates BR signaling pathway. T-DNA insertion mutant siz1-2 shows BL (Brassinolide, the most active BR) hypersensitivity and BRZ (Brassinazole, a BR biosynthesis inhibitor) insensitivity during hypocotyl elongation. In addition, expression of BES1-dependent BR-response genes is hyper-regulated in siz1-2 seedlings. The siz1-2bes1-D double mutant exhibits longer hypocotyl than bes1-D. Moreover, SIZ1 physically interacts with BES1 in vivo and in vitro and mediates the sumoylation of BES1. A K302R substitution in BES1 blocks its sumoylation mediated by SIZ1 in plants, indicating that K302 is the principal site for SUMO conjugation. Consistently, we find that sumoylation inhibits BES1 protein stability and activity. Taken together, our data show that the sumoylation of BES1 via SIZ1 negatively regulates the BR signaling pathway.

Introduction

Brassinosteroids (BRs) are a class of plant growth-promoting hormones that are structurally related to animal and insect steroid hormones (Geuns 1978, Clouse et al. 1996). BRs unlike animal steroids whose receptors are located in the nucleus that can directly bind to nuclear receptors to activate target genes (Mangelsdorf and Evans 1995), which are perceived by the plasma membrane-localized receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE 1) (Li and Chory 1997, Friedrichsen et al. 2000). Biochemical studies have revealed that BR binding to BRI1 activates its kinase activity (Wang et al. 2001), thus initiates a signal transduction cascade to activate the downstream transcription factors BES1 and BRASSINAZOLERESISTANT 1 (BZR1), which play crucial roles in regulating the expression of BR-responsive genes (Yang et al. 2011, Guo et al. 2013).

The bes1-D, caused by a substitution from proline to leucine in the PEST domain of BES1, is identified as a dominant suppressor of a weak bri1 mutant and displays constitutive BR-response phenotype (Yin et al. 2002). The bzr1-D, caused by the same method as the bes1-D, has a similar phenotype to bes1-D in the dark (Wang et al. 2002). Stability of BES1/BZR1 increased both in the bes1-D and bzr1-D mutants, which leads to constitutive BR responses, indicating that the regulation of BES1/BZR1 protein stability is vitally important during BR regulating plant growth and development.

Several studies have shown that the stability and activity of BES1/BZR1 can be regulated by post-translational modifications, including phosphorylation and ubiquitination. First of all, BIN2 (BRASSINOSTEROID-INSENSITIVE 2), a GSK3/SHAGGY-like kinase, phosphorylates BES1/BZR1 which leads to BES1/BZR1 instability and degradation (Li and Nam 2002, Yin et al. 2002, Zhao et al. 2002), whereas PP2A (protein phosphatase 2A) dephosphorylates and activates BZR1 (Tang et al. 2011). COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a dark-activated ubiquitin ligase, degrades the dark-dependent phosphorylated (inactive) form of BZR1 (Kim et al. 2014). Moreover, MAX2, a subunit of an E3 ligase, degrades both phosphorylated and dephosphorylated BES1 (Wang et al. 2013). A recent study shows that SINATs, ubiquitin E3 ligases, specifically interact with dephosphorylated BES1 and mediate its ubiquitination and degradation (Yang et al. 2017). In addition, DSK2 (DOMINANT SUPPRESSOR OF KAR 2), an ubiquitin receptor protein, interacts with BES1 and recruits BES1 to the autophagy pathway for ubiquitin-mediated BES1 degradation (Nolan et al. 2017). Therefore, it is important to illustrate the post-translational modifications of BES1 for understanding the roles of BES1 in various developmental and environmental conditions.

Sumoylation, the process of SUMO covalently conjugating to substrate proteins, is a reversible post-translational modification, similar to ubiquitination, which consists of three biochemical steps that include E1 SUMO activation enzyme, E2 SUMO conjugation enzyme, and E3 SUMO ligase (Sampson et al. 2001, Wilkinson and Henley 2010). SUMO modification in plants has been implicated in several basic aspects of cellular functions, including stress and defense responses (Kurepa et al. 2003, Catala et al. 2007, Lee et al. 2007, Miura et al. 2007, Saleh et al. 2015), hormone responses (Lois et al. 2003, Miura et al. 2009, Zheng et al. 2012, Kim et al. 2015), the regulation of flowering (Murtas et al. 2003), nutrient homeostasis (Miura et al. 2005, Park et al. 2011), photomorphogenesis (Lin et al. 2016) and so on. The PIAS (protein inhibitors of activated STATs)-type SUMO E3 ligase, SIZ1, is an SP-RING-finger protein that contains a SAP domain and a zinc-finger Miz domain, which is a principle SUMO E3 ligase that participates in all aspects of plant growth and development (Johnson and Gupta 2001, Kotaja et al. 2002).

In this study, we demonstrate the siz1-2 mutant displays BR hypersensitive in promoting hypocotyl elongation, implicating sumoylation in the regulation of BR signaling. SIZ1 negatively regulates the BR signaling pathway by mediating BES1 sumoylation at K302, and then degrades and inhibits BES1 activity. Therefore, our results provide a new sight into BES1 stability and activity via sumoylation.

Results

The SUMO E3 ligase SIZ1 negatively regulates BR signaling

In order to find out whether SIZ1 participates in the BR signaling pathway, and how it functions in BR responses, the T-DNA insertion allele of SIZ1, siz1-2/siz-3, was used. Although the siz1-2/siz-3 seedlings displayed shorter hypocotyl, it was more sensitive to BL and less sensitive to BRZ compared with wild type (WT) in hypocotyl elongation assays (Fig. 1A–D; Supplementary Fig. S1). These results demonstrate that SIZ1 may negatively regulate BR signaling.

Fig. 1

The SUMO E3 ligase SIZ1 negatively regulates BR signaling. (A) A representative example of the phenotype of 7-day-old light-grown WT (Col-0) and siz1-2 seedlings in the presence of different concentrations of BL. Scale bar, 1.5 mm. (B) The hypocotyl lengths of 7-day-old light-grown seedlings in the presence of different concentrations of BL. Data are the mean ± SD of 10–20 seedlings. The experiments were repeated three times with similar results. Significant differences were based on Student’s t-test (*P < 0.05; **P < 0.01), which is applied to all other experiments in this study. (C) A representative example of the phenotype of 7-day-old dark-grown WT (Col-0) and siz1-2 seedlings in the presence of different concentrations of BRZ. Scale bar, 10 mm. (D) The hypocotyl lengths of 7-day-old dark-grown seedlings in the presence of different concentrations of BRZ. Data are the mean ± SD of 10–20 seedlings. The experiments were repeated three times with similar results. (E) The relative expression of BR-responsive genes was determined by quantitative RT-PCR analysis. Ten-day-old WT (Col-0) and siz1-2 seedlings were used for this assay. Data are mean ± SD (n = 3) from one representative experiment. Relative expression was normalized to that of Actin. Three independent experiments were performed with similar relative trends.

Then, transcriptional levels of BR-responsive genes were detected in siz1-2 and WT seedlings to determine whether SIZ1 is indeed a negative regulator in the BR signaling pathway. We chose several BR-regulated marker genes to test (Yu et al. 2011). These genes were regulated in siz1-2 seedlings (Fig. 1E). It implicates that SIZ1 is a negative regulator of BR signaling.

Genetic interaction between SIZ1 and BES1

Genetic interaction between BES1 and SIZ1 was analyzed by crossing bes1-D and siz1-2 to create the double mutant siz1-2bes1-D. Although the single mutant siz1-2 displayed shorter hypocotyl, the double mutant siz1-2bes1-D showed enhanced hypocotyl elongation compared with bes1-D (Fig. 2A, B; Supplementary Fig. S2). In addition, BR-induced or repressed genes were upregulated or downregulated in siz1-2bes1-D relative to that in bes1-D seedlings (Fig. 2C). These results provide a further evidence that SIZ1 plays as a negative regulator in BES1 functions on hypocotyl elongation and regulation of BR-responsive genes.

Fig. 2

Genetic interaction between SIZ1 and BES1. (A) A representative example of the phenotype of 7-day-old WT (Col-0), siz1-2, bes1-D and siz1-2bes1-D seedlings. siz1-2 enhanced the hypocotyl length of bes1-D. Scale bar, 1.5 mm. (B) The hypocotyl lengths of WT (Col-0), siz1-2, bes1-D and siz1-2bes1-D seedlings 7 d after sowing. Data are the mean ± SD of 10–20 seedlings. The experiments were repeated three times with similar results. (C) Expression levels of BR-responsive genes as determined by quantitative RT-PCR analysis. Data are mean ± SD (n = 3) from one representative date set of three independent experiments. Relative expression was normalized to that of Actin (*P < 0.05; **P < 0.01).

SIZ1 interacts with BES1 in vivo and in vitro

To determine the mechanism how SIZ1 affects BES1 functions, we detected the interaction between SIZ1 and BES1. First, we performed the histidine pull-down assays. As shown, full-length of MBP-BES1 can be pulled down by His-SIZ1, but the MBP cannot be (Fig. 3A). Then truncated MBP-BES1 proteins were used to pull-down assays to map the regions of BES1 that interact with SIZ1, we found that the fragments lacking the amino acid 1–218 also were pulled down by His-SIZ1, but fragment lacking the amino acid 219–288 abolished the interaction with His-SIZ1 (Fig. 3B, C), indicating that the region of amino acid 219–288 in BES1 which contains the PEST domain contributes to its interaction with SIZ1. Then, we conducted yeast two-hybrid (Y2H) assay with full-length BES1 fused to the GAL4 DNA-binding domain (BD-BES1) and SIZ1 fused to the GAL4 activation domain (AD-SIZ1). The yeast strain AH109 harboring both BD-BES1 and AD-SIZ1 plasmids survived on medium lacking tryptophan, leucine, histidine and adenine, whereas no yeast cells cotransformed with negative control plasmids (AD+BD or AD-SIZ1+BD or AD+BD-BES1) were recovered (Fig. 3D), suggesting the interaction of SIZ1 with BES1 in vitro.

Fig. 3

SIZ1 interacts with BES1 in vivo and in vitro. (A) SIZ1 interacts with BES1 in vitro pull-down assays. His-SIZ1 pulled down MBP-BES1, but not MBP. MBP and MBP-BES1 proteins were detected using anti-MBP antibodies. His-SIZ1 proteins were detected with anti-His antibodies, which were used as equal loading. MBP and MBP-BES1 (Input) were showed. (B) The region of amino acid 219–288 of BES1 is required for its interaction with SIZ1. Letters a, b, c, d and e represented different truncated MBP-BES1 proteins [a, MBP-BES1 (1–335); b, MBP-BES1 (54–335); c, MBP-BES1 (129–335); d, MBP-BES1 (219–335); e, MBP-BES1 (289–335)]. a, b, c and d fragments all can be pulled down by His-SIZ1, but fragment e and MBP cannot be. MBP and all truncated MBP-BES1 proteins were detected with anti-MBP antibodies. His-SIZ1 proteins were detected with anti-His antibodies, which were used as equal loading. (C) Schematic diagram of various truncated BES1s. Numbers indicate the amino acid positions of these BES1 variants. (D) Y2H assay for interaction between SIZ1 and BES1. Although all yeast cells survived on medium lacking tryptophan and leucine (-LW), only cotransformed with both BD-BES1 and AD-SIZ1 plasmid cells survived on medium lacking tryptophan, leucine, histidine and adenine (-LWHA). The yeast cells cotransformed AD with BD, AD-SIZ1 with BD and AD with BD-BES1 were functioned as negative controls. (E) SIZ1 interacts with BES1 in the BiFC assays. Coexpression of BES1-cYFP with SIZ1-nYFP in tobacco leaves led to the reconstitution of YFP signal in the nucleus. Coexpression of BES1-cYFP with nYFP, and cYFP with SIZ1-nYFP were used as negative controls. For each panel, YFP, bright field (Bright), and merged images (Merge) were shown. Scale bars, 20 μm. (F) Coimmunoprecipitation analysis showing that SIZ1-GFP is associated with FLAG-HA-BES1. SIZ1-GFP and FLAG-HA-BES1 were transiently coexpressed in Col-0 protoplasts. Immunoprecipitated FLAG-HA-BES1 was detected with anti-HA antibody, and coimmunoprecipitated SIZ1-GFP was detected with anti-GFP antibody. Empty FLAG-HA vector (FLAG-HA) was used as a negative control.

To further confirm this result, we conducted a bimolecular fluorescence complementation (BiFC) assay. The YFP fluorescence signals were detected in the nucleus of Nicotiana benthamiana epidermal cells that coexpressed BES1-cYFP and SIZ1-nYFP (BES1-cYFP+SIZ1-nYFP), but YFP signals were not observed in BES1-cYFP+nYFP or cYFP+SIZ1-nYFP controls (Fig. 3E). Consistently, SIZ1-GFP was coimmunoprecipitated with FLAG-HA-BES1, but not with empty FLAG-HA vector (Fig. 3F). These results indicate that SIZ1 physically interacts with BES1 in plants.

We also detected the interaction between SUMO1 and BES1 in vitro and in vivo. The pull-down assay showed that SUMO1 could interact with BES1, and the region of amino acid 219–288 in BES1 contributed to its interaction with SUMO1 (Supplementary Fig. S3A). But mutation the SIM1/SIM2 in BES1 did not affect BES1 interaction with SUMO1 (Supplementary Fig. S3B, C). Consistently, SUMO1 interacted with BES1 in the BiFC assay (Supplementary Fig. S3D). These results showing that SUMO1 interacts with BES1 in vitro and in vivo. Furthermore, some key components of BR signaling like BRI1 and HAT1 did not interact with SIZ1 (Supplementary Fig. S4).

SIZ1 mediates sumoylation of BES1

The direct interaction between SIZ1 and BES1 suggested that BES1 may be a SUMO substrate. There are three potential sumoylation sites (K88, K205 and K302) in BES1 by SUMOplot (http://www.abgent.com/tool/sumoplot) analysis (Fig. 4A). To test this hypothesis, sumoylation assay was conducted to determine if SUMO-modified BES1 as described previously (Miura et al. 2005, Miura et al. 2007). After the reaction containing SUMO E1 (His-SAE1b and His-SAE2), SUMO E2 (His-SCE1), SUMO1 (His-SUMO1), SIZ1 (His-SIZ1) and substrate BES1 (MBP-BES1) incubated overnight at 30°C, higher molecular bands above original BES1 protein were detected using anti-MBP antibodies, suggesting that BES1 was a substrate of SUMO1 (Fig. 4B). The sumoylated BES1 protein could not be detected in the reaction without SUMO E1 and SUMO E2, or SUMO1 (Fig. 4B), indicating that SUMO1 modification of BES1 relies on E1 and E2. The presence of modified BES1 protein in the reaction without SIZ1 showed that the sumoylation of BES1 was independent of SIZ1 in vitro (Fig. 4B). To determine the sumoylation sites in BES1, we substituted K88, K205 or K302 for R, respectively, and performed an in vitro sumoylation assay. K302R substitution apparently reduced BES1-SUMO1 conjugation, but K88R or K205R substitutions did not (Fig. 4C). These results suggested that BES1 was a SUMO1 substrate and K302 was the probable sumoylation site. Next, we generated 355-FLAG-HA-BES1 overexpression plants BES1OX, 355-FLAG-HA-BES1^K302R transgenic plants BES1^K302ROX, and we also got siz1-2BES1OX by crossing siz1-2 and BES1OX, then sumoylation of BES1 was detected. First, we immunoprecipitated FLAG-HA-BES1 or FLAG-HA-BES1^K302R using anti-HA beads in BES1OX, siz1-2BES1OX or BES1^K302ROX, anti-SUMO1 antibody was used to detect sumoylated BES1. Sumoylated BES1 was detected in BES1OX, but not in siz1-2BES1OX and BES1^K302ROX (Fig. 4D). We also found that the sumoylation status of BES1 did not change after the BL treatment (Fig. 4E). These results suggest that SIZ1 mediates the sumoylation of BES1 at K302 residue in plants, and the sumoylation level of BES1 is unaffected by BL.

Fig. 4

SIZ1 mediates the sumoylation of BES1. (A) Amino acids sequence of BES1. The red amino acids showed predicted sumoylation sites in BES1. (B, C) In vitro sumoylation assays of BES1. BES1 proteins were modified by SUMO1 in the presence of SUMO E1, E2, SUMO1 (B). K302R substitution reduced the sumoylation of BES1 (C). Nonsumoylated or sumoylated BES1 proteins were detected using anti-MBP antibodies. Arrows indicate the nonsumoylated form of BES1 proteins, lines indicate sumoylated BES1 proteins. (D) Sumoylation of BES1 in the plant. Total proteins were extracted from 10-day-old BES1OX, siz1-2 BES1OX and BES1^K302ROX transgenic plants. Anti-HA beads were used to immunoprecipitate 355-FLAG-HA-BES1 or 355-FLAG-HA-BES1^K302R proteins in BES1OX, siz1-2BES1OX or BES1^K302ROX seedlings, and anti-BES1 antibodies were used to detect immunoprecipitated FLAG-HA-BES1 or FLAG-HA-BES1^K302R proteins, and anti-SUMO1 antibodies were used to determine sumoylated BES1 proteins. (E) The sumoylation level of BES1 was unaffected by BL. After growth on 1/2 MS plates for 10 d, 35S-FLAG-HA-BES1 overexpression transgenic plants BES1OX were treated with 1 μM BL (+) or mock solvent (−) for 2 h. The sumoylation level of BES1 was determined as described in (D).

SIZ1 destabilizes BES1

Sumoylation plays crucial roles on regulating protein stability and functional activity (Geiss-Friedlander and Melchior 2007). As SIZ1 mediates sumoylation of BES1, WT (Col-0) and siz1-2 seedlings were used to investigate the influence of sumoylation on BES1 stability. Although the transcriptional level of BES1 was same in siz1-2 as in WT (Fig. 5B), BES1 protein accumulated more in siz1-2 seedlings than in WT seedlings (Fig. 5A). Unphosphorylated BES1 accumulates in the nucleus in response to BL (Yin et al. 2002), then we checked BES1 levels in WT and siz1-2 seedlings in the presence of BL. The abundance of unphosphorylated BES1 was greater in siz1-2 seedlings than in WT seedlings (Fig. 5A, the top panel). We treated WT and siz1-2 seedlings with the protein synthesis inhibitor cycloheximide (CHX), and the result showed that BES1 proteins are more stable and much abundant whatever with/without BL in siz1-2 seedlings than in WT seedlings (Fig. 5A, the second and the last panels). These results indicate that the sumoylation of BES1 mediated by SIZ1 promotes instability and degradation of BES1.

Fig. 5

SIZ1 destabilizes BES1. (A) BES1 protein levels in WT (Col-0) and siz1-2 plants. The WT and siz1-2 seedlings were grown on 1/2 MS plates for 10 d. After 10 d, they were treated with BL (1/2 MS+BL+EtOH), CHX (1/2 MS+DMSO+CHX), or CHX+BL (1/2 MS+BL+CHX) for the indicated time, respectively. The abundance of BES1 was greater in siz1-2 plants than in WT plants. BES1 proteins were detected with anti-BES1 antibodies. Actin was used for equal loading. (B) BES1 transcript abundance determined by quantitative RT-PCR in Col-0 and siz1-2 seedlings. (C) Cell-free degradation assay showed the delayed degradation rate of MBP-BES1 in siz1-2 relative to that in WT seedlings, and MG132 inhibited the degradation of MBP-BES1 both in Col-0 and in siz1-2 plants. MBP-BES1 protein was detected with anti-MBP antibodies. (D) Quantification of MBP-BES1 protein levels in (C) using Image J software (https://imagej.nih.gov/ij/). MBP-BES1 protein levels at 0 min were defined as ‘1.0’.

To give more evidence, we compared BES1 protein levels in the WT (Col-0) and siz1-2 seedlings using a cell-free degradation assay. Recombinant MBP-BES1 protein was incubated in the total protein extracts from 10-day-old WT (Col-0) or siz1-2 seedlings with/without the carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132). MBP-BES1 degraded after indicated time both in WT and siz1-2 seedlings when incubated without MG132, but with a slower degradation rate in siz1-2 than in WT seedlings (Fig. 5C, the first and the second panels). When added the MG132, a 26S proteasome inhibitor, to the incubation, the degradation of BES1 was apparently inhibited both in WT and siz1-2 seedlings (Fig. 5C, the third and the last panels). These data indicate that the SIZ1-mediated BES1 sumoylation promotes its proteasome-dependent degradation. Then we extracted total proteins from BES1OX and BES1^K302ROX seedlings, Western blot analysis detected the degradation of FLAG-HA-BES1 proteins, the results showed that FLAG-HA-BES1^K302R protein had a slower degradation rate relative to FLAG-HA-BES1 protein (Supplementary Fig. S5), implicating that sumoylation promotes BES1 degradation.

Sumoylation inhibits BES1 activity

In order to determine whether sumoylation affects BES1 activity, we performed luciferase (LUC) reporter transactivation assays in Arabidopsis protoplasts. We selected two BR-repressed gene promoters (DWF4P and At2g45210P) and one BR-induced gene promoter (SAUR-AC1P) and fused with LUC gene to generate promoter-LUC reporter constructs. These reporter constructs were coexpressed with empty vector (FLAG-HA), WT BES1 or BES1^K302R in Col-0 protoplasts treated with MG132, and the reporter gene expression was used to evaluate BES1 transcriptional activity. While BES1 repressed the expression of DWF4P-LUC and At2g45210P-LUC reporter genes, the reporter genes expression was further reduced by BES1^K302R (Fig. 6B, C). Consistently, BES1 induced SAUR-AC1P-LUC reporter gene expression, when BES1^K302R expressed, the gene expression was induced more than BES1 (Fig. 6A). These results show that sumoylation inhibits BES1 transcriptional activity.

Fig. 6

Sumoylation inhibits BES1 activity. (A–C) LUC reporter transactivation assays showed sumoylation inhibits BES1 transcriptional activity. The vector (FLAG-HA), BES1 (FLAG-HA-BES1) or BES1^K302R (FLAG-HA-BES1^K302R) was coexpressed with the promoter-LUC reporter construct in Col-0 protoplasts treated with 30 μM MG132 for 3 h. The average and SDs were from three biological repeats. (D–F) ChIP assay showed BES1 binding activity was enhanced in siz1-2 compared with Col-0 whatever with/without MG132. ChIP with anti-BES1 antibodies, and the products were used for qPCR assays. TA3 was used as the internal control. Data are shown as mean ± SD of three independent experiments (*P < 0.05; **P < 0.01).

To further test the effect of sumoylation on the binding ability of BES1 in vivo, we performed chromatin immunoprecipitation (ChIP) assays. We immunoprecipitated BES1 protein from Col-0 and siz1-2 seedlings treated with/without MG132 with anti-BES1 antibody. Three BES1 direct targets were detected (SAUR-AC1, DWF4 and At2g45210). While BES1 bound to all the three gene promoters in Col-0 plants, the binding activity was significantly enhanced in siz1-2 whatever with/without MG132 (Fig. 6D–F), indicating that sumoylation mediated by SIZ1 negatively regulates the BES1-binding activity.

Discussion

In this study, we found a new mechanism that regulates BES1 stability and activity by sumoylation. First, SIZ1 is a negative regulator in the BR signaling pathway, in which siz1-2 showed BL-sensitive and BRZ-insensitive phenotype, and enhanced BR-responsive gene expression. Second, SIZ1 interacts with BES1 in vivo and in vitro, and SIZ1 possesses SUMO E3 ligase activity, which mediates SUMO1 conjugation to BES1 at K302 residue in plant. Finally, SIZ1-mediated BES1 sumoylation promotes BES1 degradation and inhibits BES1 activity.

Loss-of-function siz1-2 showed dwarf phenotype, including shorter hypocotyl (Figs. 1A, 2A), consistent with previous studies (Lin et al. 2016), common in plants defective in GA, auxin or BR. Plant growth-promoting hormones defection in SIZ1 mutant (Catala et al. 2007), might result in the dwarf phenotype of siz1-2 plants. In addition, DELLA accumulation because of the SLY1 sumoylation deduction in siz1-2 (Kim et al. 2015) also contributes to its dwarfism. When applied to appropriate concentration of BR, the hypocotyl of siz1-2 seedlings even elongated more compared with the WT seedlings (Fig. 1A, B), which is the contribution of amplified BR pathway.

In the study, we found SIZ1 binds to the amino acid 219–288 region of BES1 (Fig. 3B, C), which contains PEST domain [polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)]. PEST regions serve as proteolytic signals, and are responsive for protein degradation (Rechsteiner and Rogers 1996). SIZ1 binding to the PEST domain in BES1 could provide an explanation why BES1 is degraded by SIZ1-mediated sumoylation. SINAT-mediated BES1 degradation and COP1-mediated BZR1 degradation may differ from SIZ1-mediated BES1 degradation, since SINATs broadly regulate dephosphorylated BES1 degradation and COP1 regulates phosphorylated BES1 degradation (Kim et al. 2014, Yang et al. 2017). BL treatment did not affect BES1 sumoylation (Fig. 4E), this is to say, phosphorylated and dephosphorylated BES1 all could be sumoylated. Previous reports showed that phosphorylation and sumoylation work together in regulating protein activity and functions (Saleh et al. 2015). Our study finds that phosphorylation has no effect on BES1 sumoylation (Fig. 4E), and sumoylation did not affect BES1 phosphorylation, as phosphorylation status of BES1 is almost the same in the BES1OX and BES1^K302ROX (Supplementary Figs. S5, S6). It suggests other environmental or developmental signals maybe cross talk with BR signaling to modulate plant growth and development by regulating BES1 sumoylation.

We compared the BR response of Col-0, BES1OX and BES1^K302ROX lines in hypocotyl elongation, and found there was no difference in the control condition. However, BL promoted hypocotyl elongation was more sensitive in BES1^K302ROX than in BES1OX (Supplementary Fig. S7). Actually, overexpression of WT BES1 did not cause any visible phenotypes in most of the transgenic lines (Yin et al. 2002), and BES1^K302ROX lines also did not show visible phenotypes (Supplementary Fig. S7A, the top panel). We thought there were four forms of BES1 in plants, including phosphorylated/sumoylated form (the weakest activity), phosphorylated/desumoylated form, dephosphorylated/sumoylated form and dephosphorylated/desumoylated form of BES1 (the strongest activity). In the control condition, BES1^K302ROX and BES1OX transgenic plants showed similar hypocotyl length due to almost equal activity of phosphorylated/desumoylated form BES1 in BES1^K302ROX and phosphorylated/sumoylated BES1 in BES1OX (Fig. 4D;Supplementary Fig. S7). When applied appropriate BL, the forms of BES1 in BES1^K302ROX and BES1OX all existed in the dephosphorylated state, but dephosphorylated/desumoylated BES1 in BES1^K302ROX was more active than dephosphorylated/sumoylated BES1 in BES1OX (Supplementary Fig. S7). In other words, desumoylation-activated BES1 activity is dependent on dephosphorylation of BES1. When BES1 is phosphorylated, whatever BES1 is sumoylated or desumoylated, the activity of BES1 is inhibited; once BES1 is dephosphorylated, the activity of BES1 is further activated by desumoylation. So BR signaling pathway regulates BR-response genes expression almost by dephosphorylating BES1/BZR1. And other environmental and developmental signals maybe cross talk with BR signaling by BES1 sumoylation. Different modifications of the BES1 is useful for plants adaption to different growth and development conditions.

SUMO can covalently conjugate to target proteins mainly through a SUMO consensus motif [(ΨKxE/D; Ψ, a large hydrophobic amino acid residue; K, the acceptor lysine; x, any amino acid; E/D, glutamate or aspartate)] in substrate proteins, in addition to covalent conjugation to target proteins, SUMO can also noncovalently attach to proteins via SIMs (SUMO-interacting motifs; Kerscher 2007, Wilkinson and Henley 2010). NPR1 interacts with SUMO3 via a SIM in NPR1, and this SUMO3-NPR1 interaction is required for NPR1 sumoylation; NIb-SUMO3 interaction leads to covalent conjugation of SUMO3 to NIb, and the interaction is essential for NIb sumoylation (Saleh et al. 2015, Cheng et al. 2017). In our study, SUMO1 covalently conjugated to BES1 through a sumoylation motif, which is a SIZ1-dependent process (Fig. 4D); and BES1 also directly interacted with SUMO1 (Supplementary Fig. S3), but we failed to find the typical SIMs in BES1. SIZ1 has been shown to modulate gene expression in response to BR (Catala et al. 2007), in this study, we found SIZ1 and SUMO1 expression was repressed in bes1-D and induced in bri1-5 (Supplementary Fig. S8).

This discovery provides an insight into a post-translational regulatory mechanism of BES1. The broadly researched regulation of BES1 activity is mainly through phosphorylation and dephosphorylation (Yin et al. 2002, Yang et al. 2011). In this study, we found sumoylation inhibited BES1 transcriptional activity and DNA-binding capacities (Fig. 6), which showed that there were other post-translational modifications, such as sumoylation, regulated BES1 activity. Diverse regulatory mechanisms may function to regulate BR-responsive gene expression accurately by modulating BES1 activity during plant growth and development.

Materials and Methods

Plant materials and growth condition

Arabidopsis thaliana ecotype Columbia (Col-0) was used as the WT control. T-DNA insertion mutants, siz1-2 (At5g60410, SALK_065397) and siz1-3 (At5g60410, SALK_034008), were obtained from ABRC (Arabidopsis Biological Resource Center). The homozygous double mutant siz1-2bes1-D was obtained by crossing siz1-2 to bes1-D. 35S-FLAG-HA-BES1 overexpression transgenic plants BES1OX, and 355-FLAG-HA-BES1^K302R transgenic plants BES1^K302ROX were obtained by Agrobacterium-mediated floral transformation. siz1-2BES1OX plants were obtained by crossing siz1-2 and BES1OX#4. All of the plants were grown on half-strength MS (1/2 MS) plates and/or in soil under long-day conditions (16-h light/8-h dark) at 22°C.

Hypocotyl elongation assays

The hypocotyl elongation assays were carried out as previously described (Ye et al. 2017). In brief, seeds were sterilized with 70% ethanol and 0.1% Triton X-100 for 20 min, washed three times with 100% ethanol and dried on the filter papers. The dried seeds were sowed onto 1/2 MS plates with 0.6% agar powder and different concentrations of BL or BRZ. All the plates with seeds were placed at 4°C for 2 d. For BRZ response experiments, the plates were wrapped with three layers of aluminum foil after exposing to light for 10 h, and incubated under long-day conditions (16-h light/8-h dark) at 22°C for 7 d; for BL response experiments, the plates with seeds were directly incubated under long-day conditions (16-h light/8-h dark) at 22°C for 7 d. After 7 d, hypocotyls were measured using Image J software (https://imagej.nih.gov/ij/). About 10–20 seedlings were measured.

Plasmid constructs

For FLAG-HA-tagged transgenic plants, BES1 or BES1^K302R coding region sequence was cloned from WT and fused with FLAG-HA tag into the pCM1307 vector. For the BiFC assay, the constructs of the N- or C-termini of YFP used have been described previously (Yu et al. 2008). The coding regions of BES1 and SIZ1 or BES1 and SUMO1 were inserted into the YFP-C and YFP-N construct, respectively. For yeast two-hybrid assay, the coding region of BES1 was cloned into pGBKT7 containing a binding domain (BD), whereas SIZ1 was cloned into pGADT7 containing an activation domain (AD). For recombinant protein purification and His pull-down assay, SIZ1 or SUMO1 coding region was cloned into pET-28a vector, whereas BES1, truncated BES1 fragments, and different mutated BES1 were incorporated into pETMAL vector, respectively. For Co-IP assays in the Arabidopsis protoplasts, BES1 coding region was cloned into pCM1307 2X35SPTL vector fused with FLAG-HA tag, SIZ1-GFP construct was kindly provided by H.Z.

Transgenic plants

The construct of FLAG-HA-BES1 or FLAG-HA-BES1^K302R driven by 35S promoter was transformed into Agrobacterium tumefaciens (strain GV3101), which was used to transform WT plants by the floral dip method (Clough and Bent 1998). Transgenic lines were selected on 1/2 MS medium plus 50 μg/ml Hygromycin B. Transgene expression was analyzed by western blotting and quantitative RT-PCR.

Protein–protein interaction assay

For the BiFC assay, the BES1-cYFP, SIZ1-nYFP, SUMO1-nYFP, cYFP and nYFP constructs were transformed into A. tumefaciens (strain GV3101), respectively. Agrobacteria were grown in LB medium plus both 50 μg/ml Rifampin and 50 μg/ml spectinomycin. Combinations of Agrobacterium were infiltrated into young leaves of N. benthamiana and examined for YFP signals under a fluorescence microscope (Leica) 2 d after infiltration.

Yeast two-hybrid assay was performed using the Gal4-based two-hybrid system (Clontech). The AD-SIZ1 and BD-BES1 constructs were transformed into yeast strain AH109 using the lithium acetate method. Yeast cells were grown on medium lacking leucine, tryptophan (-LW). Transformants were plated on to medium lacking tryptophan, leucine, histidine and adenine (-LWHA) to test the interaction between SIZ1 and BES1.

For His pull-down assay, BES1, truncated BES1 fragments and different mutated BES1 fused with MBP were purified with amylose resin (NEB). SIZ1 and SUMO1 fused with His were purified with Ni-NTA agarose (Qiagen). His pull-down assays were performed as previously described (Yin et al. 2002). Ni-NTA agarose containing His-SIZ1 or His-SUMO1 were incubated with MBP, MBP-BES1, truncated MBP-BES1 proteins or mutated MBP-BES1 proteins in pull-down binding buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 0.2% Triton X-100]. The mixtures were rotated in a cold room for 2 h and the agarose was washed five times with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20) and boiled with 1× SDS loading buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted with anti-MBP antibodies.

For Co-IP assays in the Arabidopsis protoplasts, GFP-fused SIZ1 was cotransformed with FLAG-HA-fused BES1 or empty FLAG-HA vector into Arabidopsis protoplasts. After overnight incubation, total proteins were extracted from the protoplasts. The protein extracts were incubated with anti-HA affinity gel (Sigma, E-6779-1ML) at 4°C for 3 h. Next, the anti-HA affinity gel was washed with protein extraction buffer at least four times and mixed with SDS loading buffer for immunoblot analysis. The input proteins and immunoprecipitates were detected with either anti-HA antibodies or anti-GFP antibodies.

Gene expression analysis

Ten-day-old seedlings grown under long-day conditions were used for total RNA extraction and qRT-PCR analysis of BR-responsive gene expression. Total RNAs were extracted using the RNAprep pure Plant Kit (from Transgene Biotech Co. Ltd. of Qiagen, Beijing) according to the manufactures’ protocols and 2 µg total RNAs were converted into cDNAs using M-MLV Reverse Transcriptase Kit (Invitrogen, USA). The qRT-PCR analysis was carried out using the SYBR® Premix Ex TaqTM II (TAKARA) on a BIO-RAD CFX Connect™ Real-Time System according to the manufacturer’s instruction. Three independent experiments were performed, and three technical replicates of each experiment were performed. Actin was used as an internal control for the normalization of transcript levels (Czechowski et al. 2005).

DNA extraction

Four-week-old plants were used for DNA extraction. Ten milligrams leaves of Col-0, siz1-2, bes1-D and different lines of siz1-2bes1-D plants were harvested and ground to a fine powder in liquid nitrogen, and then added to 660 μl DNA extraction buffer (1 M Tris-HCl, pH 7.4, 0.5 M EDTA, 5 M NaCl, 20% SDS). After centrifugation at 13,000 rpm for 10 min, the supernatants were obtained and added to an equal volume of isopropanol. After 5 min at room temperature, the DNA extractions were collected by centrifuging at 12,000 rpm for 10 min, and then washed with 70% ethanol for two times. The DNA extractions were dissolved in 40–60 μl water.

In vitro sumoylation assay

For in vitro sumoylation assay, the coding regions of SAE1b, SAE2, SCE1, SIZ1 and SUMO1 were cloned into pET-28a vector, whereas BES1, and mutated BES1 (BES1^K88R, BES1^K205R and BES1^K302R) were incorporated into pETMAL vector. All of the constructs (His-SAE1b, His-SAE2, His-SCE1, His-SIZ1, His-SUMO1, MBP-BES1, MBP-BES1^K88R, MBP-BES1^K205R and MBP-BES1^K302R) were transformed into Escherichia coli BL21 (DE3), which was used to express recombinant proteins. His-tagged proteins were purified with Ni-NTA agarose (Qiagen), whereas MBP-tagged proteins were purified with amylose resin (NEB). The in vitro sumoylation assay was performed as described previously (Miura et al. 2005, Miura et al. 2007). In brief, 50 ng of His-SAE1b, 50 ng of His-SAE2, 50 ng of His-SCE1, 8 µg of His-SUMO1 and 100 ng of MBP-BES1 (or MBP-BES1^K88R, or MBP-BES1^K205R, or MBP-BES1^K302R) were incubated in 30 μl of reaction buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM ATP, 2 mM dithiothreitol (DTT) and 5 mM MgCl₂. The reactions were incubated over-night at 30°C. Proteins were separated by SDS-PAGE, and immunoblot analysis was performed using anti-MBP antibodies.

In vivo sumoylation assay

To determine the sumoylation status of BES1, total proteins were extracted in a buffer composed of 100 mM Tris-Cl (pH 7.5), 300 mM NaCl, 2 mM EDTA (pH 8.0), 1% TritonX-100 and 10% Glycerol (Yang et al. 2017). Then, the protein extracts were immunoprecipitated with anti-HA affinity gel (Sigma, E-6779-1ML) for 3 h in a cold room. Next, the anti-HA affinity gel was washed with protein extraction buffer at least four times, and the immunoprecipitated proteins were eluted with 2× SDS loading buffer for immunoblot analysis. The sumoylated form of BES1 was identified with anti-SUMO1 antibodies (Abcam, ab5316).

Immunoblotting analysis of BES1

Col-0 and siz1-2 Arabidopsis seedlings were grown on 1/2 MS medium under long-day conditions (16-h light/8-h dark) at 22°C, after 10 d, the seedlings were harvested and ground to a fine powder in liquid nitrogen, and then added to appropriate 2× SDS loading buffer (Martínez-García et al. 1999), boiled at 95°C for 8–10 min. After centrifugation at 12,000 rpm for 1 min, the supernatants containing total proteins were separated on 12% SDS-PAGE. Since BES1 and ACTIN have similar molecular weights, we separated our analyses onto two separate blots. One membrane was probed with anti-BES1 antibodies and another with anti-Actin antibodies for equal loading.

Protein extraction and cell-free degradation

Ten-day-old Col-0 and siz1-2 seedlings were harvested and ground into fine powder in liquid nitrogen. Total proteins were extracted in degradation buffer [26 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl₂, 4 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM DTT and 10 mM ATP]. The cell-free degradation assay was performed as described (Wang et al. 2009). In brief, the total protein extracts were adjusted to equal concentration with the degradation buffer determined by the Bio-Rad protein assay, and then 100 ng of recombinant MBP-BES1 protein was incubated in 40 μl extracts (containing 20 µg of total proteins) for individual assays. Exogenous MG132 was added to the extracts for the indicated time. The extracts were incubated at 22°C, and samples were taken at indicated times for determination of BES1 protein abundance by immunoblots with anti-MBP antibodies. The band intensity was quantified using ImageJ software.

Protoplast transformation and LUC reporter transactivation assays

The At2g45210 promoter (1,021 bp including 5′-UTR), DWF4 promoter (972 bp including 5′-UTR) and SAUR-AC1 promoter (697 bp including 5′-UTR ) were cloned into the pGreenII0800-LUC vector, separately, to generate the reporter constructs (At2g45210P-LUC, DWF4P-LUC and SAUR-AC1P-LUC); whereas the coding regions of BES1 and BES1^K302R were fused with the FLAG-HA tag into pCM1307 vector to generate the recombinant constructs (FLAG-HA-BES1 and FLAG-HA-BES1^K302R). These constructs were used to transform Col-0 Arabidopsis protoplasts. The protoplasts preparation and transformation were performed as described previously (Yoo et al. 2007). In brief, well-expanded leaves of 4-week-old plants were cut into 0.5–1 mm strips with a razor blade and incubated in freshly prepared enzyme solution [20 mM MES; pH 5.7, 1.5% (w/v) cellulase R10, 0.4% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2 and 20 mM KCl] at room temperature in the dark for 3–4 h. The protoplast-containing enzyme solution was diluted with an equal volume of W5 buffer (2 mM MES; pH 5.7, 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl) before filtration through a clean 75-mm nylon mesh. And then the flow-through was centrifuged at 100×g to obtain a protoplast pellet. The pellet was washed two times with W5 solution, the protoplasts were resuspended in MMG solution (4 mM MES; pH 5.7, 0.4 M mannitol and 15 mM MgCl₂) and kept at room temperature for PEG-mediated transformation. For each transformation, 10–20 µg of plasmid DNA was added to a 2-ml microfuge tube to which 100 μl of protoplasts were gently added. Then, 120 μl of PEG solution was added slowly and mixed by tapping and the mixture was incubated at room temperature for 15 min. A solution of 440 μl W5 was added to dilute the mixture, mixed by inverting the tubes and centrifuged at 100×g for 2 min at room temperature. The supernatant was removed and the protoplasts were resuspended in 1 ml of WI solution (4 mM MES; pH 5.7, 0.5 M mannitol and 20 mM KCl) and incubated overnight at room temperature. The protoplasts were treated with 30 μM MG132 for 3 h before they were resuspended and harvested by centrifugation at 100×g for 2 min, then the supernatants were removed and the protoplasts were freezed in liquid nitrogen for 1 min.

For LUC assay, 100 μl of protoplast lysis buffer was added to the frozen protoplasts and mixed with a pipette gun. After 5 min incubation on ice, 20 μl of the lysate harvested by centrifugation at 1,000×g for 2 min and 100 μl LUC mix were used to measure LUC activity.

ChIP assay

ChIP was performed as previously described (Saleh et al. 2008). In brief, 1.5 g of the 10-day-old Col-0 and siz1-2 plants treated with/without 30 μM MG132 for 3 h were harvested and cross-linked with formaldehyde. Chromatin was isolated and sonicated to generate fragments with the average size of 300 bp. Anti-BES1 antibodies were used to immunoprecipitate chromatin. The level of precipitated DNA fragments was quantified by qPCR. TA3 was used as an internal control for the normalization of transcript levels.

Primers

All primers used in this article are listed in Supplementary Table S1.

Funding

The National Natural Science Foundation of China [31570237 and 31670235]; the National Basic Research Program of China [973 Program (2015CB150100)]; the Development Project of Transgenic Crops of China [2016ZX08009-003–002]; National Key R&D Program of China [2018YFD0201100]; the Fundamental Research Funds for the Central Universities [SCU2019D013, SCU2018D006].

Disclosures

The authors have no conflicts of interest to declare.

Author notes

Li’e Zhang and Qing Han authors contributed equally to this work.