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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 is stabilized by HPY2 in vivo. Double transgenic plants containing 35S-FLC-FLAG3 and XVE-HA3-HPY2(A) or 35S-mFLC (K154R)-FLAG3 and XVE-HA3-AtSIZ1(B) were incubated in liquid medium with β-estradiol to induce HPY2 expression. After incubation for 15 h, HA3-HPY2, FLC-FLAG3, and mFLC-FLAG3 levels were assessed by Western blotting with anti-HA or anti-FLAG antibodies. Tubulin was used as a loading control. Numbers under lanes indicate relative intensities. Protein levels were normalized to a value of 1.00 for FLC or mFLC levels without inducer (“–” in both panels). RNA concentrations of FLC-FLAG3 and mFLC-FLAG3 were determined by real-time qRT-PCR using a FLAG primer and a gene-specific primer. Tubulin RNA was used as a loading control. (C) Graphical expression of Tubulin, FLC-FLAG3, and mFLC-FLAG3 transcript levels from (B). Bars indicate standard errors (n = 3).
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Figure 3: Flowering Locus C is stabilized by HPY2 in vivo. Double transgenic plants containing 35S-FLC-FLAG3 and XVE-HA3-HPY2(A) or 35S-mFLC (K154R)-FLAG3 and XVE-HA3-AtSIZ1(B) were incubated in liquid medium with β-estradiol to induce HPY2 expression. After incubation for 15 h, HA3-HPY2, FLC-FLAG3, and mFLC-FLAG3 levels were assessed by Western blotting with anti-HA or anti-FLAG antibodies. Tubulin was used as a loading control. Numbers under lanes indicate relative intensities. Protein levels were normalized to a value of 1.00 for FLC or mFLC levels without inducer (“–” in both panels). RNA concentrations of FLC-FLAG3 and mFLC-FLAG3 were determined by real-time qRT-PCR using a FLAG primer and a gene-specific primer. Tubulin RNA was used as a loading control. (C) Graphical expression of Tubulin, FLC-FLAG3, and mFLC-FLAG3 transcript levels from (B). Bars indicate standard errors (n = 3).

Mentions: Based on the strong interaction of HPY2 with FLC and the increase in FLC sumoylation as a result of HPY2 activity, we inferred that FLC stability could be modulated by HPY2. We therefore measured the effect of HPY2 on FLC levels using double transgenic plants with XVE-HA3-HPY2 and 35S-FLC-FLAG3 or with XVE-HA3-HPY2 and 35S-mFLC-FLAG3. In mFLC, the lysine at position 154 (sumoylation site) was mutated to arginine (K154R). HPY2 induction increased FLC levels by up to 2.3- and 3.6-fold in two independent transgenic plants (Figures 3A,C). However, no increase in mFLC level was seen after induction of HPY2 in two independent transgenic plants (Figures 3B,C). FLC and mFLC transcript levels were similar after induction of HPY2 expression, as determined using real-time qRT-PCR (Figures 3A,B). These data indicate that the FLC protein is stabilized by the E3 SUMO ligase activity of HPY2.


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 is stabilized by HPY2 in vivo. Double transgenic plants containing 35S-FLC-FLAG3 and XVE-HA3-HPY2(A) or 35S-mFLC (K154R)-FLAG3 and XVE-HA3-AtSIZ1(B) were incubated in liquid medium with β-estradiol to induce HPY2 expression. After incubation for 15 h, HA3-HPY2, FLC-FLAG3, and mFLC-FLAG3 levels were assessed by Western blotting with anti-HA or anti-FLAG antibodies. Tubulin was used as a loading control. Numbers under lanes indicate relative intensities. Protein levels were normalized to a value of 1.00 for FLC or mFLC levels without inducer (“–” in both panels). RNA concentrations of FLC-FLAG3 and mFLC-FLAG3 were determined by real-time qRT-PCR using a FLAG primer and a gene-specific primer. Tubulin RNA was used as a loading control. (C) Graphical expression of Tubulin, FLC-FLAG3, and mFLC-FLAG3 transcript levels from (B). Bars indicate standard errors (n = 3).
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Figure 3: Flowering Locus C is stabilized by HPY2 in vivo. Double transgenic plants containing 35S-FLC-FLAG3 and XVE-HA3-HPY2(A) or 35S-mFLC (K154R)-FLAG3 and XVE-HA3-AtSIZ1(B) were incubated in liquid medium with β-estradiol to induce HPY2 expression. After incubation for 15 h, HA3-HPY2, FLC-FLAG3, and mFLC-FLAG3 levels were assessed by Western blotting with anti-HA or anti-FLAG antibodies. Tubulin was used as a loading control. Numbers under lanes indicate relative intensities. Protein levels were normalized to a value of 1.00 for FLC or mFLC levels without inducer (“–” in both panels). RNA concentrations of FLC-FLAG3 and mFLC-FLAG3 were determined by real-time qRT-PCR using a FLAG primer and a gene-specific primer. Tubulin RNA was used as a loading control. (C) Graphical expression of Tubulin, FLC-FLAG3, and mFLC-FLAG3 transcript levels from (B). Bars indicate standard errors (n = 3).
Mentions: Based on the strong interaction of HPY2 with FLC and the increase in FLC sumoylation as a result of HPY2 activity, we inferred that FLC stability could be modulated by HPY2. We therefore measured the effect of HPY2 on FLC levels using double transgenic plants with XVE-HA3-HPY2 and 35S-FLC-FLAG3 or with XVE-HA3-HPY2 and 35S-mFLC-FLAG3. In mFLC, the lysine at position 154 (sumoylation site) was mutated to arginine (K154R). HPY2 induction increased FLC levels by up to 2.3- and 3.6-fold in two independent transgenic plants (Figures 3A,C). However, no increase in mFLC level was seen after induction of HPY2 in two independent transgenic plants (Figures 3B,C). FLC and mFLC transcript levels were similar after induction of HPY2 expression, as determined using real-time qRT-PCR (Figures 3A,B). These data indicate that the FLC protein is stabilized by the E3 SUMO ligase activity of HPY2.

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