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Degradation of MONOCULM 1 by APC/C(TAD1) regulates rice tillering.

Xu C, Wang Y, Yu Y, Duan J, Liao Z, Xiong G, Meng X, Liu G, Qian Q, Li J - Nat Commun (2012)

Bottom Line: A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield.Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited.Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield. The MONOCULM 1 (MOC1) gene is the first identified key regulator controlling rice tiller number; however, the underlying mechanism remains to be elucidated. Here we report a novel rice gene, Tillering and Dwarf 1 (TAD1), which encodes a co-activator of the anaphase-promoting complex (APC/C), a multi-subunit E3 ligase. Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited. Our study demonstrates that TAD1 interacts with MOC1, forms a complex with OsAPC10 and functions as a co-activator of APC/C to target MOC1 for degradation in a cell-cycle-dependent manner. Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture.

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TAD1 targets MOC1 in a cell-cycle phase-dependent manner.(a) Cell-free degradation assay showing the proteasome-dependent degradation of MOC1. Detection of Actin served as an internal control. (b) In vitro ubiquitination assay of immunopurified MOC1-GFP in the presence of recombinant HA–ubiquitin. The arrow represents unmodified MOC1-GFP, and the brace refers to ubiquitination-modified MOC1-GFP. (c) In vitro cell-free degradation assay showing the delayed degradation of expressed recombinant GST-MOC1 in tad1 and GST-mMOC1 in the wild type. The protein levels at different time points were detected by western blotting using the GST antibodies. (d) Dynamic degradation of MOC1-GFP in the wild-type (WT) and tad1 transgenic calli. Immunoblotting by the GFP antibodies showed the remaining protein levels of MOC1-GFP at different time points. (e) Profiles of TAD1 and MOC1 protein levels during the cell-cycle progression. The cell-cycle phase is determined by flow cytometry at different time points after release from synchronized rice suspension cells as indicated in the scheme at bottom. Black triangles refer to the TAD1 or MOC1 protein in the immunoblots, respectively.
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f5: TAD1 targets MOC1 in a cell-cycle phase-dependent manner.(a) Cell-free degradation assay showing the proteasome-dependent degradation of MOC1. Detection of Actin served as an internal control. (b) In vitro ubiquitination assay of immunopurified MOC1-GFP in the presence of recombinant HA–ubiquitin. The arrow represents unmodified MOC1-GFP, and the brace refers to ubiquitination-modified MOC1-GFP. (c) In vitro cell-free degradation assay showing the delayed degradation of expressed recombinant GST-MOC1 in tad1 and GST-mMOC1 in the wild type. The protein levels at different time points were detected by western blotting using the GST antibodies. (d) Dynamic degradation of MOC1-GFP in the wild-type (WT) and tad1 transgenic calli. Immunoblotting by the GFP antibodies showed the remaining protein levels of MOC1-GFP at different time points. (e) Profiles of TAD1 and MOC1 protein levels during the cell-cycle progression. The cell-cycle phase is determined by flow cytometry at different time points after release from synchronized rice suspension cells as indicated in the scheme at bottom. Black triangles refer to the TAD1 or MOC1 protein in the immunoblots, respectively.

Mentions: Cdh1 has been shown to act as a co-activator of APC/C and to function through recruiting substrates to APC/C for degradation7. The fact that TAD1 interacts with MOC1 and MOC1 is accumulated in the tad1 mutant plant suggests that MOC1 may be the target of TAD1 for degradation in rice. We therefore performed several experiments to test this possibility. We examined the degradation of endogenous MOC1 in rice plant extracts using a cell-free degradation system, which is an effective in vitro degradation assay system supporting the proteasome-mediated pathway4041. To analyse protein degradation, the cell-free degradation buffer consisting of components required for proteasome-mediated degradation was used to extract total proteins from plants. The degradation dynamics of plant proteins in the extracts were monitored by immunoblotting. As shown in Fig. 5a, when MOC1 protein levels in rice suspension cells were detected upon treatment with AEBSF (serine proteases inhibitor), Pepstatin A (acid protease inhibitor) or MG132 (proteasome inhibitor), respectively, the degradation of MOC1 was exclusively inhibited by the treatment with MG132, indicating that MOC1 was degraded in a proteasome-dependent manner. Next, we performed an in vitro ubiquitination assay using MOC1-GFP transgenic calli. The result showed that the endogenous MOC1 protein could be polyubiquitinated (Fig. 5b). Finally, we examined the degradation of expressed recombinant MOC1 monitored by the cell-free degradation assay. GST-MOC1 proteins were expressed and purified from Escherichia coli, and then added to the wild-type and tad1 extracts. The results indicated that the degradation rate of MOC1 is slower in the tad1 mutant than in the wild-type plant extracts (Fig. 5c). Consistently, when the MOC1 proteins containing a mutated D-box motif were incubated with wild-type extracts, the degradation of MOC1 was attenuated, showing that MOC1 is degraded in a D-box-dependent manner (Fig. 5c). Moreover, when MOC1 was overexpressed in the wild-type or tad1 mutant cultured cells, respectively, the dynamic degradation of MOC1 was altered in tad1. In the wild-type background, the MOC1 was degraded after 30 min and to a very low level after 60 min, but in the tad1 background it underwent only a slight degradation (Fig. 5d). Collectively, these results demonstrated that MOC1 is directly targeted by TAD1 as a substrate for degradation in vivo and its D-box at the N-terminus functions as an APC/CTAD1 recognition motif for degradation.


Degradation of MONOCULM 1 by APC/C(TAD1) regulates rice tillering.

Xu C, Wang Y, Yu Y, Duan J, Liao Z, Xiong G, Meng X, Liu G, Qian Q, Li J - Nat Commun (2012)

TAD1 targets MOC1 in a cell-cycle phase-dependent manner.(a) Cell-free degradation assay showing the proteasome-dependent degradation of MOC1. Detection of Actin served as an internal control. (b) In vitro ubiquitination assay of immunopurified MOC1-GFP in the presence of recombinant HA–ubiquitin. The arrow represents unmodified MOC1-GFP, and the brace refers to ubiquitination-modified MOC1-GFP. (c) In vitro cell-free degradation assay showing the delayed degradation of expressed recombinant GST-MOC1 in tad1 and GST-mMOC1 in the wild type. The protein levels at different time points were detected by western blotting using the GST antibodies. (d) Dynamic degradation of MOC1-GFP in the wild-type (WT) and tad1 transgenic calli. Immunoblotting by the GFP antibodies showed the remaining protein levels of MOC1-GFP at different time points. (e) Profiles of TAD1 and MOC1 protein levels during the cell-cycle progression. The cell-cycle phase is determined by flow cytometry at different time points after release from synchronized rice suspension cells as indicated in the scheme at bottom. Black triangles refer to the TAD1 or MOC1 protein in the immunoblots, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316885&req=5

f5: TAD1 targets MOC1 in a cell-cycle phase-dependent manner.(a) Cell-free degradation assay showing the proteasome-dependent degradation of MOC1. Detection of Actin served as an internal control. (b) In vitro ubiquitination assay of immunopurified MOC1-GFP in the presence of recombinant HA–ubiquitin. The arrow represents unmodified MOC1-GFP, and the brace refers to ubiquitination-modified MOC1-GFP. (c) In vitro cell-free degradation assay showing the delayed degradation of expressed recombinant GST-MOC1 in tad1 and GST-mMOC1 in the wild type. The protein levels at different time points were detected by western blotting using the GST antibodies. (d) Dynamic degradation of MOC1-GFP in the wild-type (WT) and tad1 transgenic calli. Immunoblotting by the GFP antibodies showed the remaining protein levels of MOC1-GFP at different time points. (e) Profiles of TAD1 and MOC1 protein levels during the cell-cycle progression. The cell-cycle phase is determined by flow cytometry at different time points after release from synchronized rice suspension cells as indicated in the scheme at bottom. Black triangles refer to the TAD1 or MOC1 protein in the immunoblots, respectively.
Mentions: Cdh1 has been shown to act as a co-activator of APC/C and to function through recruiting substrates to APC/C for degradation7. The fact that TAD1 interacts with MOC1 and MOC1 is accumulated in the tad1 mutant plant suggests that MOC1 may be the target of TAD1 for degradation in rice. We therefore performed several experiments to test this possibility. We examined the degradation of endogenous MOC1 in rice plant extracts using a cell-free degradation system, which is an effective in vitro degradation assay system supporting the proteasome-mediated pathway4041. To analyse protein degradation, the cell-free degradation buffer consisting of components required for proteasome-mediated degradation was used to extract total proteins from plants. The degradation dynamics of plant proteins in the extracts were monitored by immunoblotting. As shown in Fig. 5a, when MOC1 protein levels in rice suspension cells were detected upon treatment with AEBSF (serine proteases inhibitor), Pepstatin A (acid protease inhibitor) or MG132 (proteasome inhibitor), respectively, the degradation of MOC1 was exclusively inhibited by the treatment with MG132, indicating that MOC1 was degraded in a proteasome-dependent manner. Next, we performed an in vitro ubiquitination assay using MOC1-GFP transgenic calli. The result showed that the endogenous MOC1 protein could be polyubiquitinated (Fig. 5b). Finally, we examined the degradation of expressed recombinant MOC1 monitored by the cell-free degradation assay. GST-MOC1 proteins were expressed and purified from Escherichia coli, and then added to the wild-type and tad1 extracts. The results indicated that the degradation rate of MOC1 is slower in the tad1 mutant than in the wild-type plant extracts (Fig. 5c). Consistently, when the MOC1 proteins containing a mutated D-box motif were incubated with wild-type extracts, the degradation of MOC1 was attenuated, showing that MOC1 is degraded in a D-box-dependent manner (Fig. 5c). Moreover, when MOC1 was overexpressed in the wild-type or tad1 mutant cultured cells, respectively, the dynamic degradation of MOC1 was altered in tad1. In the wild-type background, the MOC1 was degraded after 30 min and to a very low level after 60 min, but in the tad1 background it underwent only a slight degradation (Fig. 5d). Collectively, these results demonstrated that MOC1 is directly targeted by TAD1 as a substrate for degradation in vivo and its D-box at the N-terminus functions as an APC/CTAD1 recognition motif for degradation.

Bottom Line: A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield.Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited.Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield. The MONOCULM 1 (MOC1) gene is the first identified key regulator controlling rice tiller number; however, the underlying mechanism remains to be elucidated. Here we report a novel rice gene, Tillering and Dwarf 1 (TAD1), which encodes a co-activator of the anaphase-promoting complex (APC/C), a multi-subunit E3 ligase. Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited. Our study demonstrates that TAD1 interacts with MOC1, forms a complex with OsAPC10 and functions as a co-activator of APC/C to target MOC1 for degradation in a cell-cycle-dependent manner. Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture.

Show MeSH
Related in: MedlinePlus