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Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response.

Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW, Valverde F, Coupland G - EMBO J. (2008)

Bottom Line: Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells.However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism.Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.

ABSTRACT
The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

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In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments.
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f3: In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments.

Mentions: The observation that the ubiquitin ligase COP1 represses CO-mediated activation of FT suggested that CO might be a substrate for COP1. To test this hypothesis, we first investigated whether COP1 was able to physically interact with CO. In the yeast two-hybrid system, we detected no interaction between CO and COP1, although an interaction between COP1 and the CO-related protein CO-LIKE3 (COL3) was previously detected by this method (Datta et al, 2006), and we were able to confirm this interaction. Therefore, whether COP1 and CO interact in vitro was tested using a co-immunoprecipitation assay (Figure 3). COP1 attached to the GAL4 activation domain (GAD:COP1) and CO were made in an in vitro transcription/translation system and combined. GAD:COP1 was precipitated with anti-GAD antibody and CO was co-precipitated with GAD:COP1 (Figure 3). In contrast, CO was not co-immunoprecipitated with GAD alone. These experiments suggest that CO interacts with COP1 in the GAD:COP1 fusion protein. Fragments of CO were also combined with GAD:COP1 to determine which regions of CO are required for the interaction with COP1. Two segments of CO were tested: one containing the region between amino acids 107 and 373, which was called COΔB-box because it did not contain the zinc-finger B-boxes found at the N terminus of CO, and a second containing the region between amino acids 1 and 331, which was named COΔCCT, because the CCT domain near the C terminus of CO was removed. In vitro precipitation experiments demonstrated that COΔB-box was co-immunoprecipitated with GAD:COP1, whereas COΔCCT was not. Therefore, the N-terminal region containing the B-boxes is not required for interaction with COP1, suggesting that the interaction with COP1 is mediated by the C-terminal region of CO that contains the CCT domain.


Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response.

Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW, Valverde F, Coupland G - EMBO J. (2008)

In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2291449&req=5

f3: In vitro interaction between CO and COP1 detected by co-immunoprecipitation. 35S-methionine-labeled CO, COΔB-box or COΔCCT was incubated with 35S-methionine-labeled GAD:COP1 or GAD and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions and pellet fractions were resolved by SDS–PAGE and visualized by autoradiography using a phosphorimager. Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD:COP1 or GAD. Error bars denote the standard error of the mean of two replicate experiments.
Mentions: The observation that the ubiquitin ligase COP1 represses CO-mediated activation of FT suggested that CO might be a substrate for COP1. To test this hypothesis, we first investigated whether COP1 was able to physically interact with CO. In the yeast two-hybrid system, we detected no interaction between CO and COP1, although an interaction between COP1 and the CO-related protein CO-LIKE3 (COL3) was previously detected by this method (Datta et al, 2006), and we were able to confirm this interaction. Therefore, whether COP1 and CO interact in vitro was tested using a co-immunoprecipitation assay (Figure 3). COP1 attached to the GAL4 activation domain (GAD:COP1) and CO were made in an in vitro transcription/translation system and combined. GAD:COP1 was precipitated with anti-GAD antibody and CO was co-precipitated with GAD:COP1 (Figure 3). In contrast, CO was not co-immunoprecipitated with GAD alone. These experiments suggest that CO interacts with COP1 in the GAD:COP1 fusion protein. Fragments of CO were also combined with GAD:COP1 to determine which regions of CO are required for the interaction with COP1. Two segments of CO were tested: one containing the region between amino acids 107 and 373, which was called COΔB-box because it did not contain the zinc-finger B-boxes found at the N terminus of CO, and a second containing the region between amino acids 1 and 331, which was named COΔCCT, because the CCT domain near the C terminus of CO was removed. In vitro precipitation experiments demonstrated that COΔB-box was co-immunoprecipitated with GAD:COP1, whereas COΔCCT was not. Therefore, the N-terminal region containing the B-boxes is not required for interaction with COP1, suggesting that the interaction with COP1 is mediated by the C-terminal region of CO that contains the CCT domain.

Bottom Line: Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells.However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism.Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.

ABSTRACT
The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

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