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First published online August 13, 2008; 10.1104/pp.108.125294 Plant Physiology 148:843-855 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Rice ROOT ARCHITECTURE ASSOCIATED1 Binds the Proteasome Subunit RPT4 and Is Degraded in a D-Box and Proteasome-Dependent Manner1,[W],[OA]Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China (Y.H., H.C., J.J., Y.X., X.W., Z.W., Z.X., K.C.); State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, China (J.D., M.Y.); Department Plant Biology, Carnegie Institution of Washington, and Department of Biological Sciences, Stanford University, Stanford, California 94305 (Z.W.); National Research Center for Plant Gene, Beijing 100093, China (Z.X., K.C.); and Graduate School of the Chinese Academy of Sciences, Beijing 100049, China (Y.H., H.C.)
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.
The development of multicellular organisms, such as plants, relies on the temporal and spatial control of cell proliferation and growth. The root tip has been widely used to study plant cell proliferation and cell growth due to its relatively simple structure and distinct regions of meristem, elongation, and mature zones. In the mitotic cell cycle program, DNA replication of the S phase is followed by the M phase, a segregation of the duplicated genetic materials into two daughter cells. Preparation gap phases, G1 and G2, precede the S and M phases, respectively.
Several genes are involved in phase transition during the cell cycle. The sequential and transient activation of cyclin-dependent kinases (CDKs) dictates a unidirectional progress through the cell cycle. Although CDKs have been identified to be the key mediators that control basic cell cycle development (Inze and De Veylder, 2006
The ubiquitin-proteasome pathway is known to control root development in Arabidopsis (Smalle et al., 2002
RPT4/SUG2 plays essential roles during the entire life of yeast cells. Deletion of the gene causes cell death (Ferrell et al., 2000
Previous studies have suggested that rice ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1), a small GTP-binding protein in plants, is involved in rice root development (Ge et al., 2004 In this study, we investigated the mechanisms of OsRAA1 in regulating rice root development using a set of biochemical and cytological approaches. An OsRAA1-interacting protein was obtained by a yeast two-hybrid screen. The in vitro and in vivo interactions between OsRAA1 and this protein were confirmed using bimolecular fluorescence complementation (BiFC), pull-down, and coimmunoprecipitation analyses. Transgenic rice plants, as well as yeast, were used to identify cell cycle regulation. A proteasome inhibition experiment revealed that the 26S proteasome is involved in the cell cycle and OsRAA1 degradation. These results support the hypothesis that the degradation of OsRAA1 is essential for the onset of anaphase in the cell cycle during root development.
OsRAA1 Interaction Protein Screening To investigate the biochemical functions of OsRAA1, a yeast two-hybrid approach utilizing OsRAA1 as a bait was conducted to identify the interaction proteins of OsRAA1. Our results indicated that the OsRAA1-binding domain (BD) fusion protein alone could not activate the expression of the reporter gene in the presence of the GAL4-activation domain (AD) vector, which indicates that OsRAA1 does not interact with GAL4-AD and does not activate transcription autonomously (data not shown). A cDNA expression library containing about 3 x 106 transformants was screened. A total of 81 colonies were obtained on a medium lacking His, Trp, and Leu but supplemented with 5 mM 3-amino-1,2,4-triazole. Among them, 32 colonies can activate the lacZ reporter gene. These colonies were able to grow on medium lacking Ade, His, Trp, and Leu. Sequence analysis revealed that they encode 18 independent fragments. Three fragments were further confirmed by transforming OsRAA1-BD back into the AH109 yeast strain, with BD used as a control. Other fragments were recognized as false positives. Homology analysis revealed three putative partner proteins: OsRPT4, chain A of isocitrate lyase, and kinesin-related protein (Fig. 1A ). The frequency of OsRPT4 was about 10% in the 32 colonies (data not shown). A fragment of a kinesin-related protein interacted with OsRAA1. Further studies on the interaction mechanism are required to interpret why the full-length kinesin shows an indistinct interaction.
To determine the binding region of OsRAA1 with RPT4, two truncated versions of BD at both the N and C termini of OsRAA1 were constructed. As shown in Figure 1B, RPT4 interacted with the C terminus rather than the N terminus of OsRAA1. The screened cDNA of OsRPT4 was 1,625 bp with an open reading frame (ORF) of 1,203 bp (Supplemental Fig. S1). The gene encodes a predicted product of 400 amino acid polypeptides of 44.6 kD, pI 7.25. Semiquantitative reverse transcription (RT)-PCR analysis indicated that the gene is expressed in all tissues tested (Supplemental Fig. S2). To determine the region of OsRPT4 that interacts with OsRAA1, we cloned the full-length form and various deleted forms of OsRPT4 into the AD construct. OsRAA1 interacted with the fragments of OsRPT4 containing amino acids 1 to 167 and 168 to 313, which possess the conserved motifs A and B, respectively, but not with the fragment containing amino acids 314 to 400 at the C terminus (Fig. 1C).
The direct interaction of OsRAA1 and OsRPT4 was further examined by pull-down, immunoprecipitation, and BiFC techniques. After incubating His-RPT4 with glutathione S-transferase (GST)-OsRAA1 or GST alone, the eluted protein complexes from glutathione-Sepharose 4B beads were examined by SDS-PAGE and western-blot analysis against antibodies specific for OsRAA1, RPT4, and GST. His-RPT4 was pulled down with GST-OsRAA1 but not with GST, which indicates that OsRAA1 and OsRPT4 directly bind to each other in vitro (Fig. 2A ).
Coimmunoprecipitation was performed to further verify the interaction between OsRAA1 and OsRPT4 in planta. Our results indicated that RPT4 can be coimmunoprecipitated with OsRAA1 using antibodies against OsRAA1 (Fig. 2B). Similarly, in transgenic yeast cells, SscRPT4 could be coprecipitated with OsRAA1 (Fig. 2B). Transiently transformed onion (Allium cepa) epidermal layer containing the fusion protein OsRAA1-GFP was used to monitor the localization of OsRAA1. Confocal analysis using this transformed tissue indicated that the fluorescence signals are evenly distributed in the cell, similar to the pattern of GFP alone (Fig. 2C). In addition, the BiFC technique was also used to demonstrate the interaction between OsRAA1 and OsRPT4. The pUCSPYNE-OsRAA1 vector contains a fusion of OsRAA1 at the N terminus of yellow fluorescent protein (YFP; OsRAA1-YFPN), and the pUCSPYCE-OsRPT4 vector contains a fusion of OsRPT4 at the C terminus of YFP (OsRPT4-YFPC). The two recombinant DNA constructs were introduced into Chinese cabbage (Brassica campestris pekinensis) epidermal cells by particle bombardment. Strong fluorescence signals were observed with the OsRAA1-YFPN and OsRPT4-YFPC combinations but were not observed when coexpressed with empty vector (Fig. 2D). The interaction of OsRAA1 and OsRPT4 showed cytoplasmic and nuclear localization, which was consistent with the OsRAA1-GFP fusion protein localization in onion epidermal cells (Fig. 2C).
Transgenic rice overexpressing OsRAA1 with increased expression at the protein level (Fig. 3A
) clearly showed suppressed growth of the primary roots in comparison with those of wild-type plants (Ge et al., 2004
Immunofluorescence analysis of microtubules during mitosis showed no abnormal phenotypes in transgenic rice compared with those of the wild type. However, OsRAA1-overexpressing rice plants showed 46% of cells localized in metaphase, whereas wild-type rice plants showed only about 28%. In addition, the proportion of cells in anaphase was lower in transgenic rice than in the wild type (12% versus 19%; Fig. 4 ). The proportion of cells in preprophase was comparable between transgenic and wild-type rice, but that in telophase was lower in transgenic rice (Fig. 4). Thus, the mitosis process was blocked to a certain extent in the transition from metaphase to anaphase in transgenic rice.
To understand the localization of OsRAA1 during cell mitosis, we stably transformed a construct harboring the OsRAA1-GFP fusion gene, as well as GFP alone, into tobacco BY-2 cells. Immunoassay of a cell line with GFP alone showed immunofluorescence in the whole cell during the cell cycle (Fig. 5A ). In contrast, in the OsRAA1-GFP transgenic line, OsRAA1-GFP was enriched at spindles, including tubulins, from metaphase to anaphase during cell division (Fig. 5B), which suggests that OsRAA1 may function at the transition from metaphase to anaphase during cell division.
To confirm the function of OsRAA1 in the cell cycle, we studied the cell system of the fission yeast Schizosaccharomyces pombe. Amino acid sequence alignment showed high identity (71%) between OsRPT4 and S. pombe RPT4 and 99% identity between the conserved motifs A and B (Supplemental Fig. S1). A BLAST search revealed no homolog of OsRAA1 in S. pombe. The constructs of the OsRAA1 overexpression vector, as well as the control vector pESPM, were transformed into yeast cells of the Leu– strain SPQ-01, and the transgenic yeast was identified subsequently by RT-PCR (Supplemental Fig. S3). The NMT1 promoter inducible by thiamine removal controls the production of OsRAA1. The immunoprecipitation results showed that OsRAA1 interacted with yeast RPT4 (Fig. 2B). Cells transformed with OsRAA1 showed reduced growth when thiamine was absent (Fig. 6Ab ), although they grew normally on medium supplemented with thiamine (Fig. 6Aa). Strains with OsRAA1 overexpressed, however, exhibited abnormal cells (Fig. 6B, c–g), whereas all cells transformed with empty vector as a control segregated normally (Fig. 6B, a and b). The growth of more than half of cells (52%) in transgenic strains was blocked at metaphase (Fig. 6Bd). These results further confirmed that overexpression of OsRAA1 causes metaphase arrest and reduced cell growth.
Degradation of OsRAA1 Depends on the Proteasome The interaction of OsRAA1 with OsRPT4 suggests that OsRAA1 is likely to be involved in regulating protein degradation. To further investigate the possible involvement of the 26S proteasome in OsRAA1-mediated root development, we examined the effects of the specific 26S proteasome inhibitors MG132 (Z-Leu-Leu-Leu-al) and MG115 (Z-leu-leu-Norvalinal) on mitosis of both wild-type and OsRAA1-overexpressing transgenic rice plants. In the presence of MG132, mitosis of the wild type showed metaphase arrest, with 47% of cells localized in metaphase compared with 28% of wild-type cells in the absence of MG132 (Fig. 4). Similarly, overexpressed OsRAA1 transgenic plants with MG132 possessed substantially more metaphase cells (62%) than those without MG132 (46%). As a control, the solvent dimethyl sulfoxide (DMSO) alone produced no effect on mitotic progression (data not shown). Thus, the activity of the 26S proteasome is likely involved in separating sister chromatids in OsRAA1-overexpressing rice; we confirmed this concept with MG115 treatment, showing 48% of metaphase cells in the wild type and 61% of such cells in transgenic rice (Fig. 4). To study the effect of proteasome inhibitors on OsRAA1 stability in vitro, we incubated protein extracts with MG132 or MG115 for 8 h and used the antiserum of OsRAA1 for the detection of the protein. After incubation, the amount of OsRAA1 in the wild type was clearly decreased and severely disrupted with the addition of His-ubiquitin and ATP (Fig. 7A ). OsRAA1 level was increased with the addition of MG132 or MG115. DMSO treatment had no effect on protein level (Fig. 7A). These results suggested that the protein level of OsRAA1 was regulated by the 26S proteasome. Similarly, in OsRAA1-overexpressing rice, OsRAA1 level was slightly reduced after incubation, and its presence was significantly disrupted by the addition of ubiquitin and ATP (Fig. 7A). OsRAA1 level in vivo was gradually reduced within 23 h of treatment with cycloheximide (CHX), an inhibitor of protein synthesis (Fig. 7, B and C). The calculated half-life was about 5 h. The half-life of OsRAA1 was extended when seedlings were treated with both MG115 and CHX (Fig. 7, B and C). With MG132 or MG115 treatment, the disruption of OsRAA1 expression was abolished, which was analogous to such treatment in the wild type. Similarly, MG132 and MG115 treatment suppressed the degradation reaction in OsRAA1-overexpressing transgenic yeast cells (Fig. 8A ).
The specific E3, APC, recognizes the key proteins in the transition from metaphase to anaphase, which are subsequently ubiquitinated on multiple sites and are degraded by the 26S proteasome (Murray, 1995  ; Cohen-Fix et al., 1996; Funabiki et al., 1996; King et al., 1996; Juang et al., 1997; Michaelis et al., 1997; Townsley and Ruderman, 1998). The common motif of the APC substrate is the D-box, a nine-amino acid sequence [RxxLxx(L/I)xN]. OsRAA1 possesses the D-box motif RGSLDLISL at amino acids 73 to 81, with R and L conserved and essential for its function. Therefore, we transformed yeast cells with mutations of Arg-73 to Gly-73 and Leu-76 to Val-76 in the D-box. With the mutated D-box, the level of OsRAA1 did not differ between nonubiquitinated controls and cells treated with inhibitors (Fig. 8B). Similarly, transgenic yeast cells with the mutated OsRAA1 did not differ in phenotype from those with the empty vector (Fig. 6Bh). The mutated OsRAA1 was not degraded by yeast 26S proteasome. Thus, these results suggest that the disruption of OsRAA1 depends on the ubiquitin-proteasome pathway.
OsRAA1 Regulates Primary Root Development by Modulating Mitosis
In Arabidopsis, several genes controlling division and differentiation in root growth and development have been identified through mutation genetics. The Arabidopsis root mutants scarecrow (scr) and short root (shr) cause reduced primary root length by degenerated cell division (Scheres et al., 1995
The expression pattern of OsRAA1 is always in rapidly growing cells, such as primordia, meristem, and the division zone of the root apex. Overexpression of OsRAA1 results in sterile florets and longer leaves at the last stage of plant development, which might be caused by both the extension of cells and increased cell numbers (Ge et al., 2004
Yeast two-hybrid analysis, together with pull-down assay, showed that OsRPT4 is a partner of OsRAA1, with the N terminus of OsRAA1 and the C terminus of RPT4 free from the interaction. Immunoprecipitation in rice, as well as BiFC data, revealed OsRAA1 interacting with OsRPT4 in vivo. The interaction was in the cytoplasm and nucleus, which is consistent with the localization of OsRAA1 alone (Fig. 2C). RPT4, a member of a very conserved AAA ATPase family, is a component of the 19S proteasome cap (Russell et al., 1996
The exact function of RPT4 and its mechanism have not been reported in plants, although some other 19S complex subunits have been studied. RPT2a is essential for maintaining meristems in Arabidopsis (Ueda et al., 2004
Cytoskeletal motors, such as the kinesin family, are involved in spindle morphogenesis at the transition from metaphase to anaphase in mitosis in yeast, animals, and plants (Lee and Liu, 2004
The different RPT4 subunit in the 19S RP might serve to recognize a distinct substrate, and a working model has been proposed (Rechsteiner et al., 1993
Other results of RPT4 in yeast suggest a role for RPT4 in degrading OsRAA1. Yeast cell division in the temperature-sensitive mutant rpt4td ceased when cells were transferred at a restriction temperature, because the SPB cannot be duplicated in the mutant cells, although the cytoplasmic microtubule showed the normal phenotype (McDonald and Byers, 1997
The destruction of key proteins by the ubiquitin/26S proteasome pathway in the transition from metaphase to anaphase is an important event in the cell cycle. The ubiquitin pathway is conserved in eukaryotic organisms and is involved in modulating the cell cycle (Hochstrasser, 1995
The cell mitosis phenotype of root tips in our transgenic plants was sensitive to the specific proteasome inhibitors MG132 and MG115. Mitosis in the wild type was blocked to a certain extent in metaphase on incubation with the inhibitors (Fig. 4), which is consistent with the results of similarly treated tobacco BY-2 cells (Genschik et al., 1998
The substrate degraded by proteasome was conjugated with ubiquitin by E1, E2, and E3 enzymes in the proteolytic process. APC acts as a specific E3 enzyme for the degradation of proteins regulating mitosis (King et al., 1996
The D-box RxxLxx(L/I)xN, as the common motif, is conserved in APC substrates (Plesse et al., 1998
Taken together, our results show that OsRAA1 modulates mitosis in rice, and this mitotic instability is required for the onset of anaphase, which is similar to the effect of the destruction of cyclins. In transgenic rice with overexpressed OsRAA1, the increased accumulation of OsRAA1 exceeded the regulation ability of the 26S proteasome and caused metaphase arrest and reduced growth of primary roots. In rice, OsRAA1 induced by auxin (Ge et al., 2004 In this study, we have shown that OsRAA1 functions in the transition from metaphase to anaphase during cell division, thus having an important role in anaphase onset during mitosis in plants. The disruption of OsRAA1 mediated by the ubiquitin pathway was required for the onset of anaphase in mitosis during root development. OsRAA1 was degraded by the 26S proteasome through interaction with OsRPT4, and APC perhaps serves to recognize OsRAA1 as E3.
Plant Materials and Yeast Strains
Rice (Oryza sativa japonica ‘Zhonghua 10’) and overexpressed OsRAA1 transgenic rice lines were described previously (Ge et al., 2004
Yeast two-hybrid screening for proteins interacting with OsRAA1 involved the use of the Matchmaker system (Clontech). The ORF of OsRAA1 was amplified by PCR with the primers 5'-ATCGAATTCATGTCAGGGGTTTGGGTGTTCAAG-3' and 5'-TATCTGCAGGTCAATTTAGGCGTCGACGACGCG-3' and the restriction sites EcoRI and PstI and then constructed in frame into the BD cloning vector pGBKT7 to act as bait. pGBKT7-OsRAA1 was transformed into AH109 cells by the lithium acetate-mediated method. Total RNA of rice seedling roots was isolated by use of the Qiagen RNeasy plant mini kit, and the cDNA library was constructed and screened according to the manufacturer's instructions (Clontech). The yeast colonies were put on plates in medium-stringency medium in the absence of Trp, Leu, and His with 5 mM 3-aminotriazol and were tested on high-stringency medium SD/–Ade–His–Leu–Trp. Colonies showing a positive signal were subsequently examined by activating the lacZ reporter gene.
The bait of OsRAA1 with the N or C terminus was generated by PCR and cloned into pGBKT7. The deletion of RPT4 was generated by PCR, and the fragments encoding amino acids 1 to 167, 168 to 313, and 314 to 400 were amplified and cloned into pGADT7.
GST and GST-OsRAA1 fusion protein were purified as described previously (Han et al., 2005
Immunoprecipitation involved the use of the IP-50 kit (Sigma-Aldrich). Approximately 100 µg of protein extracts from roots was incubated for 8 h with constant rotation at 4°C with 3 µL of the antiserum of OsRAA1 or preimmune serum. At the end of the incubation, 50 µL of protein G-Sepharose was added to immunoprecipitate the proteins for another 8-h incubation at 4°C; the precipitate was washed five times with a solution of 0.5 M NaCl and 50 mM Tris-HCl (pH 8.0), and the proteins were loaded onto polyacrylamide gels of 12% T, 3% C and 16.5% T, 3% C. T denotes the total concentration of both monomers (acrylamide and bisacrylamide), and C denotes the concentration of the cross-linker relative to the total concentration T. After gel electrophoresis, proteins were assayed with the use of anti-RPT4 antibody or antiserum of OsRAA1.
Tricine-SDS-PAGE gel electrophoresis was performed as described (Schagger and von Jagow, 1987
The PCR product of the OsRAA1 gene was ligated with XbaI and KpnI double-digested pGFP221 to create pGFP-OsRAA1, in which the cDNA region covering an ORF of OsRAA1 was fused to the 5' terminus of the GFP gene in frame, under the control of the cauliflower mosaic virus 35S promoter. Cells in the epidermal layers of onion (Allium cepa) bulbs were transformed by particle bombardment as described previously (Han et al., 2005
The interaction of OsRAA1 and OsRPT4 identified by BiFC was analyzed as described (Walter et al., 2004
The seeds of wild-type and OsRAA1-overexpressed transgenic rice plants were generated and grown for 3 to 4 d at 28°C. In each treatment, 60 root tips of 1 to 2 mm were fixed in 4% paraformaldehyde in PEM buffer (50 mM PIPES, 5 mM EGTA, 0.1 mM EDTA, and 5 mM MgSO4, pH 6.9) for 1 h, rinsed three times with PEM buffer for 10 min each, followed by 1% cellulase and 0.25% pectolyase digestion at room temperature for 50 min. After being washed with PEM and twice with 1x PBS, cells were fixed on coverslips with 1% polylysine and squashed with the use of a pencil eraser between two coverslips, then rested at –20°C for 5 min and at room temperature for 2 min. The coverslips were blocked by 1% BSA for 20 min, then incubated with 1% Triton X-100 for 30 min and rinsed three times with 1x PBS for 10 min each. The primary antibody anti-β-tubulin and the secondary antibody anti-mouse IgG fluorescein isothiocyanate conjugate were applied at room temperature for 2 h, then cells were washed three times with 1x PBS. Cells were labeled with the DNA dye 4,6-diamidino-2-phenylindole (DAPI) at 0.25 µg mL–1 in 1x PBS for 5 min. The coverslips were then sealed with nail polish. Cells were viewed with a Zeiss microscope.
BY-2 cells were maintained in Murashige and Skoog (MS) medium containing 0.5 mg L–1 2,4-dichlorophenoxyacetic acid and 30 g L–1 Suc, pH 5.8. Cells were incubated at 28°C in a shaker set at 130 rpm and subcultured weekly. The construct p35S::GFP or p35S::OsRAA1-GFP within a binary vector (pBI121) was introduced into Agrobacterium tumefaciens (strain C58) by electroporation and used to transform BY-2 cells. Transfected BY-2 cells were transferred onto MS medium containing kanamycin (100 µg mL–1) and cefotaxim (200 µg mL–1) for 3 to 4 weeks until transformed colonies were formed. Resistant cell colonies were identified by GFP fluorescence with a fluorescence microscope, and more than five positive cell lines were further transferred into MS liquid medium containing the antibiotics mentioned to initiate suspension culture and used for subsequent analysis.
Coimmunolocalization of OsRAA1 and tubulin in transgenic BY-2 cell lines was as described (Li et al., 2006
Rice seedlings at 3 d old were treated with a solution of 100 µM CHX in the presence or absence of 100 µM MG132. Proteins were then extracted from the seedlings at 0, 5, 10, 15, 20, and 23 h followed by western-blot analysis. Probes were antibodies to OsRAA1, and the antibodies also cross-reacted with a smaller nonspecific band, which served as a loading control. Western blots were quantified by densitometry using the BioCapt program. OsRAA1 band intensity was normalized to the loading control band and then normalized to the time zero controls. Each decrease of 1 unit of log2 (band intensity) is equivalent to one half-life (Stommel and Wahl, 2004
The procedure was as described (Yamamoto et al., 2004
The ORF of OsRAA1 was constructed in a vector of pESPM with an NMT1 promoter, which was inducible with thiamine removal. The transformants were selected on plates containing minimal medium with thiamine at 30°C. Cells from the selected clones were grown to midexponential phase in minimal medium containing thiamine at 30°C, washed three times with the minimal medium without thiamine, and then incubated at 30°C for 22 h. Then, cells were examined with a light microscope. The transformant cells of pESPM plasmid were used as a control. Cells were fixed with methanol or formaldehyde and stained with DAPI dissolved in 1x PBS (1 µg mL–1) to visualize the DNA, then examined with a Zeiss microscope.
Protein was extracted by the use of the Yeast Buster reagent (Novagen). The yeast cells were harvested from 100 mL of liquid culture (optical density at 600 nm up to 0.8) by centrifugation at 3,000g for 10 min at 4°C. Yeast Buster reagent and THP solution were added to the cell pellet and incubated on a shaking platform at 45°C for 40 min. After centrifugation at 16,000g for 20 min at 4°C, the supernatant was obtained as a protein mixture. The degradation assay of OsRAA1 in yeast followed that of rice as described above. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY659938.
The following materials are available in the online version of this article.
We thank Dr. Stephen Albert Johnston (Center for Biomedical Inventions, University of Texas-Southwestern Medical Center) for the generous gifts of antibodies against RPT4 and Dr. Jorg Kudla (Institut fur Botanik und Botanischer Garten, Universitat Munster) for the kind gifts of plasmids for BiFC assay. Received June 24, 2008; accepted August 9, 2008; published August 13, 2008.
1 This work was supported by the Major State Basic Research Program of the People's Republic of China (grant no. 2005CB120806) and the National Science Foundation of China for Distinguished Young Scholars (grant no. 30525026), as well as by innovation grants from the Chinese Academy of Sciences. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kang Chong (chongk{at}ibcas.ac.cn).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.125294 * Corresponding author; e-mail chongk{at}ibcas.ac.cn.
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(catalytic) and β (regulatory). A dominant-negative mutant of CK2 stably expressed in tobacco (Nicotiana tabacum) BY-2 cells interrupted the G1/S and G2 phases (Moreno-Romero et al., 2008
