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Arabidopsis HIGH PLOIDY2 Sumoylates and Stabilizes Flowering Locus C through Its E3 Ligase Activity.

Kwak JS, Son GH, Kim SI, Song JT, Seo HS - Front Plant Sci (2016)

Bottom Line: Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC.In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation.These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University Seoul, South Korea.

ABSTRACT
Flowering Locus C (FLC), a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC. In vitro and vivo pull-down assays showed that FLC physically interacts with HPY2. In vitro assays showed that the stimulation of FLC sumoylation by HPY2 was dependent on SUMO-activating enzyme E1 and -conjugating enzyme E2, indicating that HPY2 was an E3 SUMO ligase for FLC. In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation. Flowering time in hpy2-2 mutants was shorter than in wild-type plants under long- and short-day conditions, with a greater effect under short-day conditions, and FLC was downregulated in hpy2-2 mutants. These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

No MeSH data available.


Related in: MedlinePlus

Flowering Locus C (FLC) is sumoylated by HPY2 in vitro.Arabidopsis His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, GST-HPY2, His6-AtSUMO1, and GST-FLC-Myc were overexpressed in E. coli and purified with Ni2+-NTA or glutathione affinity columns as appropriate. (A) Sumoylation of GST-FLC-Myc was assayed in the presence or absence of E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1. FLC sumoylation was detected by Western blotting with anti-Myc antibody. (B) To identify the sumoylation site on FLC, GST-FLCm1-Myc (K54R), GST-FLCm2-Myc (K135R), and GST-FLCm3-Myc (K154R) were overexpressed in E. coli and purified using a glutathione affinity column. The reaction mixture contained E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1 without (-) or with (+) a mutant protein instead of GST-FLC-Myc. FLC sumoylation was detected by Western blotting with anti-Myc antibody.
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Figure 2: Flowering Locus C (FLC) is sumoylated by HPY2 in vitro.Arabidopsis His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, GST-HPY2, His6-AtSUMO1, and GST-FLC-Myc were overexpressed in E. coli and purified with Ni2+-NTA or glutathione affinity columns as appropriate. (A) Sumoylation of GST-FLC-Myc was assayed in the presence or absence of E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1. FLC sumoylation was detected by Western blotting with anti-Myc antibody. (B) To identify the sumoylation site on FLC, GST-FLCm1-Myc (K54R), GST-FLCm2-Myc (K135R), and GST-FLCm3-Myc (K154R) were overexpressed in E. coli and purified using a glutathione affinity column. The reaction mixture contained E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1 without (-) or with (+) a mutant protein instead of GST-FLC-Myc. FLC sumoylation was detected by Western blotting with anti-Myc antibody.

Mentions: The strong interaction between HPY2 and FLC (Figures 1B,C) suggested that FLC could be modified by SUMO through E3 SUMO ligase activity of HPY2. Therefore, we next tested whether HPY2 had E3 SUMO ligase activity for FLC. In vitro sumoylation reactions were performed using His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, His6-AtSUMO1, GST-HPY2, and GST-FLC-Myc. The amount of sumoylated GST-FLC-Myc was increased by HPY2 in an E1- and E2-dependent manner (Figure 2A).


Arabidopsis HIGH PLOIDY2 Sumoylates and Stabilizes Flowering Locus C through Its E3 Ligase Activity.

Kwak JS, Son GH, Kim SI, Song JT, Seo HS - Front Plant Sci (2016)

Flowering Locus C (FLC) is sumoylated by HPY2 in vitro.Arabidopsis His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, GST-HPY2, His6-AtSUMO1, and GST-FLC-Myc were overexpressed in E. coli and purified with Ni2+-NTA or glutathione affinity columns as appropriate. (A) Sumoylation of GST-FLC-Myc was assayed in the presence or absence of E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1. FLC sumoylation was detected by Western blotting with anti-Myc antibody. (B) To identify the sumoylation site on FLC, GST-FLCm1-Myc (K54R), GST-FLCm2-Myc (K135R), and GST-FLCm3-Myc (K154R) were overexpressed in E. coli and purified using a glutathione affinity column. The reaction mixture contained E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1 without (-) or with (+) a mutant protein instead of GST-FLC-Myc. FLC sumoylation was detected by Western blotting with anti-Myc antibody.
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Figure 2: Flowering Locus C (FLC) is sumoylated by HPY2 in vitro.Arabidopsis His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, GST-HPY2, His6-AtSUMO1, and GST-FLC-Myc were overexpressed in E. coli and purified with Ni2+-NTA or glutathione affinity columns as appropriate. (A) Sumoylation of GST-FLC-Myc was assayed in the presence or absence of E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1. FLC sumoylation was detected by Western blotting with anti-Myc antibody. (B) To identify the sumoylation site on FLC, GST-FLCm1-Myc (K54R), GST-FLCm2-Myc (K135R), and GST-FLCm3-Myc (K154R) were overexpressed in E. coli and purified using a glutathione affinity column. The reaction mixture contained E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-AtSCE1), E3 (GST-HPY2), and His6-AtSUMO1 without (-) or with (+) a mutant protein instead of GST-FLC-Myc. FLC sumoylation was detected by Western blotting with anti-Myc antibody.
Mentions: The strong interaction between HPY2 and FLC (Figures 1B,C) suggested that FLC could be modified by SUMO through E3 SUMO ligase activity of HPY2. Therefore, we next tested whether HPY2 had E3 SUMO ligase activity for FLC. In vitro sumoylation reactions were performed using His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, His6-AtSUMO1, GST-HPY2, and GST-FLC-Myc. The amount of sumoylated GST-FLC-Myc was increased by HPY2 in an E1- and E2-dependent manner (Figure 2A).

Bottom Line: Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC.In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation.These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University Seoul, South Korea.

ABSTRACT
Flowering Locus C (FLC), a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC. In vitro and vivo pull-down assays showed that FLC physically interacts with HPY2. In vitro assays showed that the stimulation of FLC sumoylation by HPY2 was dependent on SUMO-activating enzyme E1 and -conjugating enzyme E2, indicating that HPY2 was an E3 SUMO ligase for FLC. In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation. Flowering time in hpy2-2 mutants was shorter than in wild-type plants under long- and short-day conditions, with a greater effect under short-day conditions, and FLC was downregulated in hpy2-2 mutants. These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

No MeSH data available.


Related in: MedlinePlus