Plant and Cell Physiology Advance Access originally published online on February 23, 2008
Plant and Cell Physiology 2008 49(3):420-432; doi:10.1093/pcp/pcn019
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Identification of Dynamin as an Interactor of Rice GIGANTEA by Tandem Affinity Purification (TAP)
1Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan
*Corresponding author: E-mail, simamoto{at}bs.naist.jp; Fax, +81-743-72-5502.
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Abstract |
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GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants.
Keywords: Flowering - GI - Proteomics - Rice - Tandem affinity purification (TAP)
Abbreviations: ADL, Arabidopsis dynamin-like protein; CaM, calmodulin; CaMV, cauliflower mosaic virus; CBP, calmodulin-binding peptide; CCA1, CIRCADIAN CLOCK ASSOCIATED 1; CDF1, CYCLING DOF FACTOR 1; CO, CONSTANS, ELF4, EARLY FLOWERING 4; FKF1, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1; FT, FLOWERING LOCUS T; GFP, green fluorescent protein; GI, GIGANTEA; GIS, GLABROUS INFLORESCENCE STEM; LD, long days; LDP, long-day plant; LHY, LATE ELONGATED HYPOCOTYL; LUX, LUX ARRHYTHMO; PCL1, PHYTOCLOCK 1; RT–PCR, reverse transcription–PCR; SD, short days; SDP, short-day plant; SPY, SPINDLY; TAP, tandem affinity purification; TEV, tobacco etch virus; TOC1, TIMING OF CAB EXPRESSION 1; YFP, yellow fluorescent protein; ZTL, ZEITLUPE.
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Introduction |
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The transition from vegetative to reproductive development is one of the most important steps in the lives of plants. This transition is regulated by genetic as well as environmental factors such as light and temperature (Boss et al. 2004
, Imaizumi and Kay 2006, Kobayashi and Weigel 2007). The photoperiod or day-length is one of the most important factors in the determination of flowering time. Plants are able to measure changes in day-length and use this information to flower during the optimal season. Plants can be classified into three groups depending on their responses to the photoperiod: long-day plants (LDPs), which promote flowering under long-day (LD) conditions, short-day plants (SDPs), which flower under short-day (SD) conditions, and day-neutral plants, which flower independently of the photoperiod.
A number of genes that play important roles in the determination of flowering time were identified in Arabidpsis, an LDP (Koornneef et al. 1998, Yanovsky and Kay 2003, Searle and Coupland 2004). Flowering time genes can be classified into three groups based on their primary functions. The first group contains genes involved in the circadian clock. They include CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), EARLY FLOWERING 4 (ELF4) and LUX ARRHYTHMO (LUX)/PHYTOCLOCK 1 (PCL1) (Schaffer et al. 1998, Wang and Tobin 1998, Strayer et al. 2000, Alabadi et al. 2001, Doyle et al. 2002, Hazen et al. 2005, Onai and Ishiura 2005). CCA1 and LHY encode myb-like transcription factors which bind specifically to the cis-element termed the ‘morning element’ in the promoter of TOC1 and LUX/PCL1, and repress expression of TOC1 (Harmer et al. 2000) and LUX/PCL1 (Alabadi et al. 2001, Hazen et al. 2005) during the morning hours. TOC1, ELF4 and LUX/PCL1 activate the expression of CCA1 and LHY (Alabadi et al. 2001, Doyle et al. 2002, Hazen et al. 2005). Post-translational modification is one of the important mechanisms for the regulation of flowering time, and the phosphorylation of CCA1, and the degradation of TOC1 by the clock-associated F-box protein (ZTL) have been demonstrated.
The second group of flowering time genes in Arabidopsis contains the photoreceptors. They include phytochromes encoded by PHYA and PHYB and cryptochromes encoded by CRY1 and CRY2 (Lin 2000a, Lin 2000b). Photoreceptors perceive various light stimuli and regulate expression of flowering time genes, or alter the stability of proteins encoded by flowering time genes. The third group contains genes which are specifically involved in regulation of the other flowering time genes. They include GIGANTEA (GI) (Fowler et al. 1999, Park et al. 1999), CONSTANS (CO) (Putterill et al. 1995, Suarez-Lopez et al. 2001) and FLOWERING LOCUS T (FT) (Kardailsky et al. 1999, Kobayashi et al. 1999). Transcription of these genes is regulated by the circadian clock. GI has been shown to regulate flowering and the circadian clock pathway in Arabidopsis, and has been identified in many other plant species (Suarez-Lopez et al. 2001, Mizoguchi et al. 2005). The GI protein interacts with the F-box protein FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) (Nelson et al. 2000) in a blue light-dependent manner, and regulates the stability of CYCLING DOF FACTOR 1 (CDF1), a repressor of CO expression (Imaizumi et al. 2005, Sawa et al. 2007). GI also regulates TOC1 expression, and it regulates the stability of ZTL by interacting with it (Kim et al. 2007).
CO, together with a HAP protein complex which has a CCAAT-box-binding function, regulates FT expression (Ben-Naim et al. 2006, Wenkel et al. 2006, Cai et al. 2007). FT is expressed only in the phloem of the leaf, but the protein FT interacts with FD, a transcriptional factor in the shoot apical meristem, and the FT–FD complex activates expression of the floral organ identity gene AP1 (Abe et al. 2005, Wigge et al. 2005). Recently it was shown that proteins encoded by the Arabidopsis gene FT and its rice homolog Hd3a act as florigens, or mobile flowering signals (Corbesier et al. 2007, Jaeger and Wigge 2007, Mathieu et al. 2007, Tamaki et al. 2007). The rice orthologs of GI, CO and FT have been identified as OsGI, Hd1 and Hd3a, respectively (Yano et al. 2000, Hayama et al. 2002, Kojima et al. 2002). Under SD conditions OsGI activates Hd1 expression and Hd1 induces Hd3a expression, while under LD conditions, Hd1 represses Hd3a expression (Hayama et al. 2003). In Arabidopsis, the regulation of FT expression by CO is reversed under LD conditions. Therefore, the difference in the regulation of FT by CO in Arabidopsis and rice was proposed to be the molecular mechanism that explains the difference between LDPs and SDPs (Hayama et al. 2003).
Comprehensive analyses of protein–protein interactions have become increasingly important for biological studies in the post-genome era. Large-scale yeast two-hybrid screening has been used for this purpose (Ito et al. 2000, Uetz et al. 2000). However, more effective methods for analyzing protein–protein interactions under native conditions need to be developed. Improvements in mass spectrometry combined with the development of various protein purification technologies have made it possible to analyze proteins that are present at very low levels in their native states. Therefore, current limitations in the analysis of protein–protein interactions are not in the purification methods available, but in the methods for identifying proteins involved in the interactions. The tandem affinity purification (TAP) method was developed for the high yield purification of protein complexes formed under native conditions (Rigaut et al. 1999, Puig et al. 2001). The TAP method uses transgenes expressing N- or C-terminal-fused TAP-tagged versions of the target proteins. The protein complex is isolated from transgenic cells by affinity purification using the TAP tag. The TAP tag consists of two IgG-binding domains of Staphylococcus aureus protein A and a calmodulin (CaM)-binding peptide (CBP), separated by a tobacco etch virus (TEV) protease cleavage site (Rigaut et al. 1999). In their original study, Rigaut et al. (1999) used this method to identify the U1 snRNP, involved in pre-mRNA splicing, and Mak 3/10/31, which is involved in protein modifications. The TAP method was originally developed in yeast and has since been used in mammalian cells (Knuesel et al. 2003), insects (Forler et al. 2003) and Escherichia coli (Gully et al. 2003). However, only a few studies of plant protein interactions using the TAP method have been reported (Rohila et al. 2004, Rubio et al. 2005, Rohila et al. 2006, Van Leene et al. 2007). There have been no reports on the purification and identification of novel protein complexes using the TAP method in plants.
In order to study the functions of the rice GI protein, we attempted to isolate OsGI-interacting proteins from cultured rice cells expressing a TAP-tagged OsGI protein. We were able to purify seven OsGI-interacting proteins by this method. Moreover, we confirmed the interaction of OsGI with one of these proteins, dynamin, using co-immunoprecipitation. We also characterized Arabidopsis plants with mutations in the dynamin genes ADL3 and ADL6.
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Results |
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Generation of transgenic rice plants and cultured cells expressing a TAP-tagged OsGI protein
To purify OsGI-interacting proteins, transgenic rice plants and cultured cells expressing TAP-tagged versions of the OsGI protein were generated. A full-length OsGI cDNA was cloned into each of two binary vectors (Nakagawa et al. 2007
) by the LR reaction. These binary vectors were designed to produce recombinant proteins carrying the TAP tag at either the N- or C-terminal ends. Expression of the recombinant genes was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The TAP tag used in this study was the same as the original one used by Rigaut et al. (1999), and consisted of the protein A gene, the TEV protease recognition site and the CBP. The resulting two vectors, encoding modified versions of OsGI in which the TAP tag was fused at either the N-terminus (35S::TAP:OsGI, SGN) or the C-terminus (35S::OsGI:TAP, SGC) (Fig. 1A), were introduced into Agrobacterium tumefaciens for rice transformation. Transgenic rice plants and cultured cell lines were generated. Since the TAP method has not been successfully used to isolate novel plant proteins and it is not easy to analyze proteins in rice leaves, we thought that cultured rice cells might be suitable material to apply TAP for rice. We were able to detect TAP-tagged OsGI transcripts by reverse transcription–PCR (RT–PCR) in both he SGN and SGC cell lines (Fig. 1B). These lines were used for further experiments.
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Western blot analysis of TAP-tagged OsGI in transgenic rice cells lines
To detect the OsGI protein by Western blotting, we produced a polyclonal OsGI antibody using the N terminal (–3 to + 865) part of the OsGI sequence. The Arabidpsis GI protein was previously shown to localize to the nucleus, by transient assays using β-glucuronidase (GUS) or green fluorescent protein (GFP) fusion proteins (Huq et al. 2000
). The intracellular localization of the rice protein OsGI has not been examined previously. Therefore, we used our OsGI antibody to analyze proteins in cytosolic and nuclear fractions isolated from transgenic cell lines by Western blotting. The OsGI protein was detected in both the cytosolic and the nuclear fractions of wild-type and transgenic cell lines, and showed the expected size of approximately 130 kDa (Fig. 2A). An antibody against histone H3 was used as a control for the nuclear-localized proteins. Interestingly, we observed repeatedly that the concentration of the OsGI in the cytosol was higher than that in the nucleus (Fig. 2A). In addition to the endogenous OsGI, the TAP-tagged OsGI (
150 kDa) was also detected in the transgenic SGC line, which carried the C-terminal TAP-tagged construct. Even though the transcript levels were similar in both cell lines (Fig. 1B), the TAP-tagged OsGI protein was not detected in the SGN line, carrying the N-terminal construct (Figs. 1A, 2A). The reason for the lack of protein expression in the SGN line is not clear at this stage. The SGC line was used for purification of the OsGI-interacting proteins, and in other experiments. To investigate further the intracellular localization of OsGI, a 35S::OsGI:GFP construct was produced and introduced into rice leaf sheath cells by particle bombardment (Fig. 2B–D). A Ubi::YFP vector, which promotes expression of the yellow fluorescent protein (YFP) in both the cytosol and the nucleus, was used as a control. Fluorescence signals were observed using a confocal laser scanning microscope 12 h after introduction of the vectors. Both the GFP and YFP signals were detected in both the cytosol and the nucleus (Fig. 2B–D). These results were consistent with those of the Western blot analysis and it was concluded that the OsGI protein was localized in both the cytosol and the nucleus in rice cells.
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-OsGI) was used for the detection of TAP-tagged OsGI (open arrowhead) and OsGI (filled arrowhead, top panel) in cytosolic and nuclear extracts from wild-type and transgenic cell lines. The TAP-tagged OsGI protein was expressed only in the SGC line. Detection of histone H3 (black arrowhead, middle panel) using the The TAP-tagged OsGI protein was functional in transgenic rice plants
Before purifying the OsGI-interacting proteins by the TAP method, we needed to examine whether the TAP-tagged OsGI protein was functional in rice plants. In a study using Arabidopsis, Rubio et al. (2005
) performed mutant complementation tests and furthermore showed that a TAP-tagged protein was incorporated into the protein complex, as was the untagged protein. Although the Tos-17 insertion lines of OsGI have recently become available, they produced few seeds (Abe et al. unpublished results), and Agrobacterium-mediated transformation of rice requires seed-derived callus. Therefore, we used another approach to examine whether the TAP-tagged OsGI protein was functional in rice plants. We have previously reported that overexpression of OsGI delayed flowering in transgenic plants compared with the wild type under both SD and LD conditions (Hayama et al. 2003). Therefore, we analyzed the flowering times of transgenic plants overexpressing the TAP-tagged OsGI under both SD and LD conditions. Wild-type and OsGI-overexpressing plants (the OX 24 line) produced by Hayama et al. (2003) were used as negative and positive controls, respectively. The SGC lines C33 and C53 showed delayed flowering compared with the wild type under SD conditions (Fig. 3A). The degree of the delay in flowering time in the SGC lines was similar to or higher than that in the OX 24 line. On the other hand, the flowering time of the SGN line N23 was similar to that of the wild type. Under LD conditions, the flowering time of each transgenic line, including OX 24, was not significantly different from that of the wild type (Fig. 3B). The reason for this observation is not known. However, these results suggest that the TAP-tagged OsGI protein (in SGC lines) was functional in transgenic rice plants.
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Purification of OsGI-interacting proteins by the TAP method
OsGI-interacting proteins were purified from SGC rice cell cultures expressing the TAP-tagged OsGI protein (referred to in this section as OsGI-TAP). In order to follow the progress of the purification, samples were taken during each step of the TAP procedure and analyzed on Western blots using both the OsGI and TAP tag antibodies (Fig. 4A). A protein extract from wild-type cultured cells was used as a negative control. In the first step, in which the protein A motif of OsGI-TAP was bound to the IgG beads, almost all of the OsGI-TAP in the solution seemed to bind to the beads (Fig. 4A, extract after IgG). Since the original TEV protease contained its own cleavage site, the efficiency of this step was not very high. Therefore, we used AcTEV (Invitrogen), which has no recognition site and is therefore more stable, for cleavage at the TEV site. After digestion with the AcTEV protease, the size of the OsGI-TAP protein was reduced by the size of the protein A peptide, which is approximately 15 kDa (Fig. 4A, IgG eluate and IgG beads). Although some of the OsGI-TAP protein remained on the IgG beads, most was cleaved by AcTEV (Fig. 4A, IgG eluate and IgG beads).
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In the second step of the purification, CaCl2 was added to the protein solution to induce CaM binding. In the flowthrough from the CaM beads, the OsGI-TAP signal was not detected, indicating that most of the OsGI-TAP bound to the beads (Fig. 4A, CaM beads flowthrough). Then EGTA was added to chelate the Ca2+, and the OsGI was eluted. Finally, a strong OsGI-TAP signal was detected in the CaM beads eluate (Fig. 4A, CaM eluate). Furthermore, the OsGI-TAP signal was not detected in a solution derived from the CaM beads, suggesting that the OsGI-TAP was completely eluted from the beads (Fig. 4A, CaM beads). The eluted proteins obtained by this purification method were separated by SDS–PAGE, and visualized by SYPRO-RUBY staining. Some protein bands were specifically found in the SGC lines and were not detected in the wild-type cells (Fig. 4B). Fluorescent staining was found to have a similar level of sensitivity to that of silver staining with glutaraldehyde. Thus, fluorescent staining was used to detect protein bands in the mass spectrometric identification of the proteins.
Identification of OsGI interacting proteins
Gels containing candidate OsGI-interacting proteins, and corresponding gels from wild-type cells, were cut into 1 mm slices. Then the proteins in each slice were extracted, analyzed by Q-TOF mass spectrometry, and identified in a MASCOT database search (Fujiwara et al. 2006). If a protein was found in gels from both transgenic and wild-type cells, we considered that it was a non-specific protein. Experiments were repeated three times, and proteins which were identified in all three experiments were considered as candidates for OsGI-interacting proteins. A total of seven proteins were identified (Table 1).
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One of the putative OsGI-interacting proteins was a dynamin homolog. There are many more isoforms of dynamin in plants compared with animals, and their functions appear to be redundant. In Arabidopsis there exists a family of proteins termed the Arabidopsis dynamin-like proteins (ADLs). There are at least five forms (A–E) of ADL1. ADL1A, ADL1C, ADL4 and ADL5 are involved in cell plate formation in dividing cells (Kang et al. 1998
, Praefcke and McMahon 2004). ADL2A carries a chloroplast transit peptide at its N-terminus, and is found in plastids (Kang et al. 1998). ADL2B is associated with mitochondria in dividing cells. ADL6 has a high level of homology with ADL3, and is the only member of the classical dynamin family. ADL6 is involved in trafficking from the trans-Golgi network to the vacuole (Jin et al. 2001).
Amongst the other putative OsGI-interacting proteins, Nup155 is associated with the formation of the nuclear envelope and pore complex (Franz et al. 2005) and is modified by O-GlcNAc (Wells et al. 2002). Another was the S2 subunit of the 26S proteasome, which has been shown in Arabidopsis to degrade the GI protein in the dark (David et al. 2006). The putative vacuolar protein sorting-associated protein (gi|50919153) has homology with VPS35, which recognizes the retrieval signal domains of cargo proteins during their recruitment to vesicles (Seaman et al. 1997, Nothwehr et al. 2000). The multifunctional protein interacts with cortical microtubules and RNA in rice (Chuong et al. 2002, Chuong et al. 2005). The human HP68 (ATP-binding protein ABCE1 or RNase L inhibitor) is a host protein required for formation of the human immunodeficiency virus, type 1 (HIV-1) (Zimmerman et al. 2002, Lingappa et al. 2006, Dooher et al. 2007). However, the functions of plant proteins with homology to HP68 are not known.
OsGI interacts with dynamin in vivo
To examine further the interactions between OsGI and the putative OsGI-interacting proteins obtained by the TAP method, rice cell lines (M11 and M12) expressing a myc-tagged OsGI protein (OsGI:myc) were generated. After immunoprecipitation with the myc antibody, the OsGI:myc protein was detected in both the M11 and M12 cell lines by Western blot analysis, using either the OsGI antibody or the myc antibody (Fig. 5A). Because the OsGI:myc signal was much stronger in the M12 cell line than in the M11 line, the M12 cell line was used for further experiments. In a Western blot analysis of proteins immunoprecipitated using the myc antibody, a specific signal was detected in the M12 line with a plant dynamin antibody (Fig. 5B). Since dynamin is highly conserved in plants, we used a commercially available antibody made using the Arabidopsis dynamin. These results indicate that OsGI interacts with dynamin in vivo.
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Phenotypic analysis of Arabidopsis dynamin mutants
To study the function of dynamin in relation to OsGI function we searched for available rice lines with insertion mutations in the putative dynamin gene. However, we were not able to obtain any rice dynamin mutants. Therefore, we searched for Arabidopsis dynamin mutants. First, to identify the closest Arabidopsis homologs of the putative rice dynamin (gi|5092565), a phylogenetic tree was created (Fig. 6). According to the tree, the rice dynamin homolog (gi|5092565) has the highest homology with ADL3 (AT1G59610) and ADL6 (AT1G10290). Three T-DNA insertion mutants in each of the Arabidopsis ADL3 and ADL6 genes were found by a database search, and used for further analysis (Fig. 7A, B).
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We first analyzed the flowering times of these mutants, and found that there were no differences in flowering time between the mutants and wild-type plants. However, the mutants exhibited an aerial rosette phenotype in the main stem (Fig. 7D–F). This phenotype has been described previously in a late flowering ecotype of Arabidopsis (Grbic and Bleecker 1996
). The frequencies of plants showing the aerial rosette phenotype varied among the insertion lines. The lines SALK_150606 and SALK_071036 showed the highest frequencies amongst the ADL3 and ADL6 mutant lines, respectively (Fig. 7F). These results indicate that the dynamin proteins, which may interact with GI in Arabidopsis, are required for the suppression of elongation in the main stem. However, the possible role of dynamin in the regulation of flowering time remains to be studied. |
Discussion |
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Intracellular localization of OsGI
In this study, the intracellular localization of the OsGI protein was analyzed by two different methods. The first method involved the fractionation of protein extracts into cytosolic and nuclear proteins by centrifugation, and Western blot analysis of the separated protein fractions using an OsGI-specific antibody. The second method was a transient expression assay using a GFP-fused OsGI protein in rice leaf sheath cells. Results from both experiments indicated that OsGI was localized in both the nucleus and the cytosol. It was previously reported that a GFP-fused Arabidopsis GI protein was detected in the nucleus of onion epidermal cells when it was transiently expressed (Huq et al. 2000
). However, the results of a recent fractionation study showed that GI is present in both the nucleus and the cytosol of Arabidopsis (Kim et al. 2007).
Recently, the Arabidopsis GI was shown to interact with ZTL in the cytosol, and this interaction increased the stability and accumulation of the GI–ZTL complex (Kim et al. 2007). Accumulation of this complex was shown to regulate a clock protein TOC1, which controls circadian clock oscillation in plants. The Arabidopsis GI was also shown to interact with FKF1 in the nucleus, and this complex regulates photoperiodic flowering by controlling the stability of CDF1, a repressor of CO transcription (Sawa et al. 2007). Therefore, GI seems to play roles in flowering and circadian clock oscillations by interacting with two different proteins in the two compartments of the cell.
Although our results indicated that OsGI was localized in both the nucleus and the cytosol, the putative OsGI-interacting proteins obtained by the TAP method in this study (Table 1) are primarily cytosolic proteins. This could be because the TAP-tagged OsGI protein was mainly located in the cytosol of the cultured rice cells used for purification (Fig. 2A). Alternatively, only strong interactors of OsGI may have been identified in this study. The use of a cross-linker described by Rohila et al. (2004) may be useful for identifying proteins which have weaker interactions with target proteins.
Use of the TAP method in plants
Rohila et al. (2004) were the first to apply the TAP method in plants. They introduced a TAP-tagged GVG (NTAPi:GVG) construct into Nicotiana benthamiana leaves by transient Agrobacterium-mediated transformation. Proteins extracted from the transgenic cells were used for purification by the TAP method, and HSP90 and HSP70 were identified as GVG-interacting proteins. This confirmed previous reports on the interaction of HSP90 and HSP70 with GVG in humans and plants. By using an optimized purification method, the same group expressed 41 TAP-tagged rice protein kinases in transgenic rice plants, recovered 36 (95%) of the TAP-tagged proteins after purification and identified interacting proteins for 23 (56%) of the kinases (Rohila et al. 2006). No further studies on the isolated proteins have been reported yet.
The efficiency of protein identification in the study described above was lower tha n that in yeast, in which endogenous proteins are replaced with the TAP-tagged proteins by homologous recombination (Rigaut et al. 1999). One possible reason for the lower efficiency of the TAP method in previous plant studies may be due to competition for interacting proteins between the untagged endogenous and tagged proteins. In our study the OsGI:TAP gene was introduced into wild-type rice; therefore, the TAP-tagged OsGI protein was likely to compete with the endogenous OsGI for interaction. This could have decreased the efficiency of the TAP method in our experiments. To circumvent this potential problem, we used the 35S promoter to achieve high levels of expression of the TAP-tagged OsGI transgenes. However, no clear quantitative difference was observed between the levels of endogenous OsGI and OsGI:TAP by Western blot analysis (Fig. 2A).
In our purification, heat shock proteins and elongation factors were identified, but they were shown to be non-specific proteins (data not shown). A similar observation was made in another study (Rohila et al. 2006). Rubio et al. (2005) performed TAP tagging experiments using CSN3, a component of the COP9 signalosome complex in Arabidopsis. They used the rhinovirus C3 protease, which is highly active at low temperatures, instead of the TEV protease. We used AcTEV to increase the efficiency of the procedure, because it has higher stability than TEV. Rubio et al. (2005) used 6x His and 9x myc repeats in place of the CBP, to avoid the use of EGTA in the final purification step. The CBP was not changed in our study because we focused on the purification of OsGI-interacting proteins. If the objective was to analyze the protein complex functions after purification, then a tag which does not require Ca2+ chelation for purification may be advantageous.
Tags might affect the functions of target proteins. In the current study, both N-terminal and C-terminal TAP fusions were produced, but the tagged protein was produced only from the C-terminal fusion (in the SGC lines). SGC plants showed delayed flowering under SD conditions but not under LD conditions. The reason for the lack of protein expression in the SGN line is not clear at this stage. OsGI-overexpressing plants were previously shown to be late flowering under both SD and LD conditions (Hayama et al. 2003). Rubio et al. (2005) generated transgenic plants expressing 31 TAP-tagged genes, including photomorphogenesis-related and 26S proteasome-related genes. These transgenic plants exhibited differences in the levels of gene expression and in their abilities to complement mutant genes (Rubio et al. 2005).
Rubio et al. (2005) and Rohila et al. (2004, 2006) performed pioneering experiments using the TAP tagging method in plants. Several other studies have also applied the TAP method in plants; however, the transgenic plants were used only for Western blot analysis of the tagged proteins or for pull-down assays. Thus far there have been no reports on the identification of novel proteins which were shown to interact with the tagged targets by this method in plants. In this study we were able to identify seven OsGI-interacting proteins, including a dynamin-like protein, by the TAP method. Furthermore, we showed that OsGI interacts with dynamin, by co-immunoprecipitation with the myc antibody from extracts of OsGI:myc cell cultures.
Possible functions of the Arabidopsis dynamin-like proteins ADL3 and ADL6 in plant development
Dynamin is a high molecular weight GTPase which plays a critical role in vesicle formation on the plasma membrane during endocytosis (Praefcke and McMahon 2004). In Arabidopsis, the dynamin-like protein family consists of at least six forms (1–6), and there are five forms of ADL1 (A–E). Of these, ADL6 shows the greatest homology with the animal dynamin 1, and has a conserved GTPase domain at the N-terminus, a pleckstrin homology domain at the center, and a proline-rich motif at the C-terminus (Jin et al. 2001). ADL6 plays a role in the formation of vesicles for trafficking from the trans-Golgi network to the vacuole (Jin et al. 2001). ADL3 is expressed weakly in most tissues, except for the siliques, in which it is highly expressed (Mikami et al. 2000). Studies of dynamin in Arabidopsis have mainly dealt with intracellular localization, and little attention has been given to protein function. In the current study, dynamin mutants were grown under LD (16 h light/8 h dark) conditions and their development was analyzed. The results indicated that the flowering times of the dynamin mutants were not different from those of the wild-type plants, but an aerial rosette phenotype was observed in the main stems of the mutants. This was a diagnostic phenotype found in the Arabidopsis ecotype Sy-0 (Grbic and Bleecker 1996). ENHANCER OF AERIAL ROSETTE (EAR) and AERIAL ROSETTE 1 (ART1) were identified as the genes required for this phenotype in the Sy-0 ecotype (Grbic and Bleecker 1996). The flowering time of Sy-0 is delayed, because ART1 activates expression of a floral repressor FLC (Poduska et al. 2003). The aerial rosette phenotype has also been observed in other Arabidopsis mutants. Overexpression of GLABROUS INFLORESCENCE STEM (GIS), a C2H2-type transcription factor, caused the aerial rosette phenotype in transgenic plants (Gan et al. 2006). The GIS gene was shown to play a role in epidermal cell differentiation during the vegetative phase. An analysis of the spy gis double mutant indicated that SPY (SPINDLY) acts upstream of GIS and suppresses GIS expression (Gan et al. 2006). These functions of SPY and GIS are related to gibberellic acid signaling (Gan et al. 2006). Therefore, it is possible that the aerial rosette phenotype was caused by changes in gibberellic acid signaling. It is also possible that the dynamin mutations analyzed in the current study may have alterations in gibberellic acid signaling. This remains to be studied.
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Materials and Methods |
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Cloning of the OsGI cDNA and plasmid construction
The OsGI cDNA was synthesized from total RNA of rice leaves. Three cDNA fragments covering three different regions of OsGI were separately amplified using three pairs of PCR primers. The primers were designed using the OsGI coding sequence, with alternations to create restriction enzyme sites. The primers used were: OsGI1-F-TOPO 5'-caccaagcttATGTCAGCTTCAAATGAG-3' OsGI1-R 5'-gaattctctagaTGGTGCCTCGAGAAGACC-3' for fragment 1, OsGI2-F 5'-TTGGTCTTCTCGAGGCAC-3' and OsGI2-R 5'-CTTCTAGAGGCTCAGCTT-3' for fragment 2, and OsGI3-F 5'-AAGCTGAGCCTCTAGAAG-3' and OsGI3-R 5'- gaattcGCAAGTGAGTGGGCAGCC-3'for fragment 3. Plasmid construction was carried out using the Gateway technology (Invitrogen, Carlsbad, CA, USA). The OsGI cDNA was inserted into the Gateway destination vector pGWB containing the TAP tag (Nakagawa et al. 2007
) using the LR recombination reaction.
Generation and screening of transgenic plants and cultured cells
Plant transformation vectors carrying the OsGI cDNA were introduced into A. tumefaciens for rice transformation. Agrobacterium-mediated transformation of rice was performed according to a published protocol (Hiei et al. 1994). Transformed callus was selected using hygromycin, and plants were regenerated from the transformed callus.
Production of the OsGI antibody
An 868 bp fragment from the 5' end of OsGI (from ATG –3 to +865) was amplified by RT–PCR using the primers NdeOsGI (F) 5'-catATGTCAGCTTCAAATGAGAAGTGGATT-3' and OsGIXhoI (R) 5'-ctcGAGGTAATAGAAGTGCAGGAACAG-3', and subcloned into the pBluescript SK+ vector (Stratagene, La Jolla, CA, USA). The fragment was re-isolated using NdeI and XhoI, and inserted into the expression vector pET-15b (Novagen, Madison, WI, USA). The resultant vector was introduced into BL21 (DE3) competent cells (Stratagene, La Jolla, CA, USA). The antibody was commercially produced (Medical and Biological Laboratories, Nagoya, Japan).
Western blot analysis
Rice cultured cells were ground to a powder in liquid nitrogen and the powder was suspended in buffer A [20 mM HEPES, 250 mM sucrose, 10% glycerol, 10% EtOH, 0.5% Triton and the complete protease inhibitor cocktail (Roche Diagnostics K.K., Tokyo, Japan)]. The suspension was homogenized using a polytron (KINEMATICA; PT-MR 3100, Bohemia, NY, USA) at 15,000 r.p.m. for 1 min on ice then filtered through four layers of miracloth (Calbiochem, Darmstadt, Germany). The filtered extract was centrifuged at 3,300xg for 10 min at 4°C and the supernatant was used as the cytosolic fraction. The pellet was resuspended in buffer B (20 mM HEPES, 10% sucrose, 10% glycerol, 2.5% EtOH, 0.5% Triton-X and the complete protease inhibitor cocktail) and centrifuged at 3,300xg for 10 min at 4°C. The pellet was resuspended in a modified buffer A, in which the glycerol concentration was changed to 35%. The suspension was transferred to a beaker and stirred on ice while 2 µl of 5 M NaCl was added every minute for 1 min to make the final concentration to 5 mM. Then the suspension was transferred to a new tube and centrifuged at 15,000 r.p.m. for 30 min at 4°C. The supernatant was used as the nuclear fraction. Protein samples were boiled in sample buffer, run on SDS–polyacrylamide gels (7.5 and 12.5%) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The histone H3 antibody (Upstate, Billerica, MA, USA) was used for detection of the histone protein.
Analysis of OsGI cellular localization by transient assays
The OsGI cDNA was subcloned into a modified binary vector carrying the GFP gene, for transient assays. The 35S::OsGI:GFP and Ubi::YFP (Wong et al. unpublished results) vectors were introduced into protoplasts from Oc cells by electroporation (Wong et al. 2004) and into rice leaf sheath cells by particle bombardment. After 12 h of incubation, GFP and YFP fluorescence was analyzed using a confocal laser scanning microscope (Zeiss; LSM510, Jena, Germany)
Purification of OsGI-interacting proteins by the TAP method
Rice cultured cells (40 g fresh weight) were ground in liquid nitrogen. TAP purification was performed essentially as described previously with minor modifications (Rigaut et al. 1999). The AcTEV protease was used instead of TEV to increase the efficiency of the purification. The eluted samples were precipitated using STRATACLEAN (Stratagene, La Jolla, CA, USA).
Identification of proteins by mass spectrometry
Mass spectometry was performed as described previously (Fujiwara et al. 2006). Gels containing the proteins to be analyzed were visualized by fluorescent staining and cut into 1 mm slices. The proteins were digested by trypsin in the gels and then identified by Q-TOF mass spectrometry (Waters, Milford, MA, USA) and a MASCOT database search of the NCBI (National Center for Biotechnology Information) database. The analyses were performed three times, and proteins that were detected all three times, only in the SGC cell line, were considered to be OsGI-interacting proteins.
Immunoprecipitation of OsGI and dynamin
The OsGI cDNA was introduced into the pGWB vector (Nakagawa et al. 2007) to fuse with the myc tag, and transgenic cell lines containing the myc-tagged OsGI protein were produced. Proteins were extracted from 1 g of cultured cells with extraction buffer (25 mM Tris–HCl pH 7.5, 5 mM EDTA, 10 mM MgCl2, 10% sucrose, 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol and the complete protease inhibitor cocktail). The protein concentration in the extract was adjusted to 2 mg ml–1, and the extract was incubated with 50 µl of protein G-Sepharose 4 Fast Flow (GE Healthcare UK Ltd, Buckinghamshire, UK). The supernatant was transferred to a new tube, 4 µl of myc antibody (Nacalai Tesque, Kyoto, Japan) were added, and the solution was incubated for 16 h at 4°C with gentle rotation. A 50 µl aliquot of protein G–Sepharose beads was added and the solution was incubated for a further 3 h with gentle rotation. After centrifugation, the beads were washed three times with well chilled extraction buffer. After removing the supernatant, 50 µl of sample buffer were added to the beads. The supernatants from the boiled beads were then subjected to SDS–PAGE. The immunoprecipitated proteins were analyzed on Western blots using a commercially available plant dynamin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Arabidopsis ADL3 (AT1G59610) and ADL6 (AT1G10290) mutants
ADL3 (AT1G59610) and ADL6 (AT1G10290) belong to the same group of dynamin-like genes as the putative rice dynamin gene (gi|50912565). Three T-DNA insertion lines were obtained for each gene from the T-DNA Express (SIGnal Arabidopsis Gene Mapping Tool). ADL3 (AT1G59610) mutants were SALK_124686, SALK_134887 and SALK_150606, and ADL6 (AT1G10290) mutants were SALK_011319, SALK_071039 and SALK_018859.
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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We thank Dr. Tsuyoshi Nakagawa at Shimane University for pGWBs. We thank members of the Plant Molecular Genetics Lab at NAIST for their comments and participation in discussions.
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2Present address: Plant Science Education Unit, Laboratory of Plant Protein Analysis, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan
3Present address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan
4Present address: Faculty of Agriculture, Iwate University, Morioka, 020-8550 Japan
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(Received December 10, 2007; Accepted February 3, 2008)
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  Compiled by, F. Tooke, T. Chiurugwi, and N. Battey Flowering Newsletter bibliography for 2008 J. Exp. Bot., June 23, 2009; (2009) erp154v1. [Full Text] [PDF]
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