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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

Interaction of HPY2 with FLC.(A) His6-FLC and GST-HPY2 were overexpressed in Escherichia coli and purified with Ni2+-NTA or glutathione affinity columns. (B) The His6-FLC protein was pulled down with the GST-HPY2 protein, separated on 11% SDS-polyacrylamide gels, and analyzed by Western blotting with anti-His antibody. (C)In vivo interaction of HPY2 and FLC. Wild-type (WT) plants were infiltrated with different combinations of 35S-Myc6-FLC or 35S-HPY2-FLAG3 constructs. Total protein was extracted from each sample and immunoprecipitated with anti-FLAG antibody. After immunoprecipitation, Myc6-FLC was detected by Western blotting with anti-Myc antibody. Myc6-FLC and HPY2-FLAG3 expression was also examined by Western blotting with anti-Myc and anti-FLAG antibodies, respectively.
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Figure 1: Interaction of HPY2 with FLC.(A) His6-FLC and GST-HPY2 were overexpressed in Escherichia coli and purified with Ni2+-NTA or glutathione affinity columns. (B) The His6-FLC protein was pulled down with the GST-HPY2 protein, separated on 11% SDS-polyacrylamide gels, and analyzed by Western blotting with anti-His antibody. (C)In vivo interaction of HPY2 and FLC. Wild-type (WT) plants were infiltrated with different combinations of 35S-Myc6-FLC or 35S-HPY2-FLAG3 constructs. Total protein was extracted from each sample and immunoprecipitated with anti-FLAG antibody. After immunoprecipitation, Myc6-FLC was detected by Western blotting with anti-Myc antibody. Myc6-FLC and HPY2-FLAG3 expression was also examined by Western blotting with anti-Myc and anti-FLAG antibodies, respectively.

Mentions: Previous results showed that E3 SUMO ligase AtSIZ1 interacted with FLC to inhibit sumoylation (Son et al., 2014). Here, we aimed to identify the E3 SUMO ligase that stimulated FLC sumoylation. HPY2, an Arabidopsis E3 SUMO ligase possessing an SP-RING (SIZ/PIAS-RING) domain, was chosen as a candidate. An in vitro pull-down assay was used to examine the interaction of HPY2 with FLC. Recombinant proteins GST-HPY2 and His6-FLC were overexpressed in E. coli and purified with glutathione or Ni2+-NTA resins (Figure 1A). The results showed that GST-HPY2 was able to pull down His6-FLC (Figure 1B). The interaction between HPY2 and FLC was also examined by immunoprecipitation (IP). Two constructs, 35S-Myc6-FLC and 35S-HPY2-FLAG3, were coinfiltrated into the leaves of WT plants. After IP with anti-FLAG antibody, Myc6-FLC was examined by Western blot analysis using anti-Myc antibody. Myc6-FLC was clearly detected (Figure 1C), indicating a strong interaction between HPY2 and FLC, consistent with the results of the in vitro pull-down experiment.


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)

Interaction of HPY2 with FLC.(A) His6-FLC and GST-HPY2 were overexpressed in Escherichia coli and purified with Ni2+-NTA or glutathione affinity columns. (B) The His6-FLC protein was pulled down with the GST-HPY2 protein, separated on 11% SDS-polyacrylamide gels, and analyzed by Western blotting with anti-His antibody. (C)In vivo interaction of HPY2 and FLC. Wild-type (WT) plants were infiltrated with different combinations of 35S-Myc6-FLC or 35S-HPY2-FLAG3 constructs. Total protein was extracted from each sample and immunoprecipitated with anti-FLAG antibody. After immunoprecipitation, Myc6-FLC was detected by Western blotting with anti-Myc antibody. Myc6-FLC and HPY2-FLAG3 expression was also examined by Western blotting with anti-Myc and anti-FLAG antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Interaction of HPY2 with FLC.(A) His6-FLC and GST-HPY2 were overexpressed in Escherichia coli and purified with Ni2+-NTA or glutathione affinity columns. (B) The His6-FLC protein was pulled down with the GST-HPY2 protein, separated on 11% SDS-polyacrylamide gels, and analyzed by Western blotting with anti-His antibody. (C)In vivo interaction of HPY2 and FLC. Wild-type (WT) plants were infiltrated with different combinations of 35S-Myc6-FLC or 35S-HPY2-FLAG3 constructs. Total protein was extracted from each sample and immunoprecipitated with anti-FLAG antibody. After immunoprecipitation, Myc6-FLC was detected by Western blotting with anti-Myc antibody. Myc6-FLC and HPY2-FLAG3 expression was also examined by Western blotting with anti-Myc and anti-FLAG antibodies, respectively.
Mentions: Previous results showed that E3 SUMO ligase AtSIZ1 interacted with FLC to inhibit sumoylation (Son et al., 2014). Here, we aimed to identify the E3 SUMO ligase that stimulated FLC sumoylation. HPY2, an Arabidopsis E3 SUMO ligase possessing an SP-RING (SIZ/PIAS-RING) domain, was chosen as a candidate. An in vitro pull-down assay was used to examine the interaction of HPY2 with FLC. Recombinant proteins GST-HPY2 and His6-FLC were overexpressed in E. coli and purified with glutathione or Ni2+-NTA resins (Figure 1A). The results showed that GST-HPY2 was able to pull down His6-FLC (Figure 1B). The interaction between HPY2 and FLC was also examined by immunoprecipitation (IP). Two constructs, 35S-Myc6-FLC and 35S-HPY2-FLAG3, were coinfiltrated into the leaves of WT plants. After IP with anti-FLAG antibody, Myc6-FLC was examined by Western blot analysis using anti-Myc antibody. Myc6-FLC was clearly detected (Figure 1C), indicating a strong interaction between HPY2 and FLC, consistent with the results of the in vitro pull-down experiment.

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