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. 2008 Oct;148(2):843-55.
doi: 10.1104/pp.108.125294. Epub 2008 Aug 13.

Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner

Ye Han  1 , Hong Cao, Jiafu Jiang, Yunyuan Xu, Jizhou Du, Xin Wang, Ming Yuan, Zhiyong Wang, Zhihong Xu, Kang Chong
Affiliations

Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner

Ye Han et al. Plant Physiol. 2008 Oct.

Abstract

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.

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Figures

Figure 1.
Figure 1.
OsRAA1 interacts with RPT4 in yeast. A, Yeast two-hybrid assay demonstrating OsRAA1 interacting with OsRPT4, partial fragment of kinesin protein, and isocitrate lyase. B, Overview of the OsRAA1 domain interacting with OsRPT4 by two-hybrid assay, with AD used as a control. OsRPT4 strongly bound to the C terminus rather than the N terminus of OsRAA1. C, Derivatives of OsRPT4 interacting with OsRAA1, with BD as a control. OsRAA1 interacted with amino acids 1 to 167 of OsRPT4 at the N terminus, amino acids 168 to 313 containing motifs A and B, but not with amino acids 314 to 400 at the C terminus.
Figure 2.
Figure 2.
Interaction of OsRAA1 with OsRPT4. A, OsRAA1 interacts with OsRPT4. GST-OsRAA1 fusion protein and GST were expressed and purified from Escherichia coli, adsorbed onto GST-Sepharose 4B, and incubated with His-OsRPT4. After being washed, the protein-bound protein was fractionated by 12% SDS-PAGE and underwent western-blot analysis with antiserum of RAA1 (lane 4), GST antibodies (lane 2), or RPT4 antibodies (lanes 3 and 5). One-tenth of the total input of the E. coli total protein inducing His-RPT4 expression was loaded onto SDS-PAGE gels and detected by RPT4 antibodies (lane 1). B, Western-blot results of in vivo coimmunoprecipitation confirming the interaction between OsRAA1 and OsRPT4. Protein extracts from transgenic rice roots (left and middle) or transgenic yeast cells (right) were immunoprecipitated with 3 μL of anti-OsRAA1 (lanes 1, 3, and 5) or control preimmune serum (lanes 2, 4, and 6). The immunoprecipitated solution then underwent 12% T and 16.5% T, 3% C SDS-PAGE, then was incubated with anti-RPT4 (1:400; lanes 1, 2, 5, and 6) and anti-OsRAA1 (1:400; lanes 3 and 4; top images). The bottom images show Coomassie Brilliant Blue staining of loading controls. Molecular mass markers are indicated at left. Stars indicate immunoglobulin heavy chains. C, Localization of OsRAA1-GFP fusion protein in onion cells. D, BiFC view of OsRAA1 and OsRPT4 interaction in Chinese cabbage epidermal cells on transient expression assay. Corresponding differential interference contrast images are shown at top. Bars = 20 μm.
Figure 3.
Figure 3.
Expression of OsRAA1 and root morphological responses in transgenic lines. A, Identification of OsRAA1 expression in transgenic line (OsRAA1-OX) and wild-type (wt) plants by western-blot analysis. Coomassie Brilliant Blue (CBB) staining of a loading control is shown at bottom. B, Identification of OsRAA1 expression in RNAi lines (Ri-L) and wild-type (wt) plants by western-blot analysis. CBB staining of a loading control is shown at bottom. C, Phenotypes (left) and statistics (right) of RNAi transgenic seedling height aboveground. Significant differences between wild-type and RNAi lines were determined by two-sample t test (* P < 0.01). D, The unchanged cell length of RNAi transgenic rice lines (left), and the cell length with no significant differences (right).
Figure 4.
Figure 4.
Effects of MG132 and MG115 on the mitotic index in immunofluorescence. Transgenic (OsRAA1-OX) and wild-type (wt) root tip cells were double stained with β-tubulin antibody, anti-mouse IgG fluorescein isothiocyanate (FITC) conjugate shown in green, and DAPI shown in blue in the presence of 100 μm MG132 or MG115. Mitotic phase is indicated at left. DIC, Differential interference contrast. Bars = 2 μm.
Figure 5.
Figure 5.
Localization of OsRAA1 during cell mitosis. Immunolocalization of OsRAA1 and tubulin in BY-2 cells stably expressing OsRAA1-GFP or GFP. Confocal images (GFP in green and β-tubulin in red) were collected from various cell phases of the cell cycle. A, Localization of GFP and tubulin as a control. Merged images are shown at bottom. B, Localization of OsRAA1-GFP and tubulin. Merged images are shown at bottom. Bars = 20 μm.
Figure 6.
Figure 6.
Phenotypic characterization of overproduced OsRAA1 yeast strains. A, Overexpressed OsRAA1 suppresses the growth rate of the yeast cells. SPQ-01 yeast cells were grown overnight at 30°C, and 0.2 × 106 cells mL−1 were inoculated into 50 mL of Edinburgh minimal medium and grown in a medium containing thiamine (a) or not (b). Growth was monitored by optical density at 600 nm (OD600). B, Chromosome segregation defects in yeast cells. Transgenic cells were grown with vigorous agitation at 30°C to log phase in the presence of thiamine. After being washed, cells were put in liquid culture with no thiamine for 24 h. A vector-transformed cell that just completed separation (a) and one in interphase (b) are shown as controls. c to g show aberrant transgenic cells, and their percentage location is 5%, 52%, 4%, 3%, and 7%, respectively. For every panel, more than 500 cells were examined. The transgenic line with mutant OsRAA1 showed a normal shape (h). Bars = 10 μm.
Figure 7.
Figure 7.
Effects of the inhibitors on OsRAA1 degradation mediated by the 26S proteasome, and half-life of OsRAA1. The mixture was separated on 16.5% T, 3% C SDS-PAGE and underwent western-blot analysis with antiserum of OsRAA1. A, Effect of the inhibitor on OsRAA1 degradation was monitored with the antibody. OsRAA1 of wild-type and transgenic rice and detected with the addition of 10 μg of ubiquitin in the presence of 100 μm MG132 or MG115 with 25°C incubation for 8 h. The columns show relative levels of OsRAA1 detected by the antibody. DMSO was used as a control. Coomassie Brilliant Blue staining of a loading control is shown at bottom. B, OsRAA1 half-life in rice seedlings. Protein synthesis was inhibited by treatment with CHX (100 μm) for the indicated times. At the same time, seedlings were treated with MG132 (100 μm). The OsRAA1 was monitored at the indicated times. Data represent results from more than three experiments. C, Quantitation of the OsRAA1 destabilization shown in B.
Figure 8.
Figure 8.
Effects of mutation in the D-box motif of OsRAA1 on its degradation by the 26S proteasome. The yeast protein extracts were separated on 16.5% T, 3% C SDS-PAGE and underwent western-blot analysis with the antibody to OsRAA1. OsRAA1 (A) and D-box mutant OsRAA1 R73G L76V (B) were detected with the addition of 10 μg of ubiquitin in the presence of 100 μm MG132 or MG115 with 25°C incubation for 8 h. The columns show relative levels of OsRAA1 detected by the antibody. DMSO was used as a control for the inhibitor treatment. Coomassie Brilliant Blue (CBB) staining of a loading control is shown at bottom.
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