Transcriptional repression by MYB 3R proteins regulates plant organ growth
2015; Springer Nature; Volume: 34; Issue: 15 Linguagem: Inglês
10.15252/embj.201490899
ISSN1460-2075
AutoresKosuke Kobayashi, Toshiya Suzuki, Eriko Iwata, Norihito Nakamichi, Takamasa Suzuki, Poyu Chen, Misato Ohtani, Takashi Ishida, Hanako Hosoya, Sabine Müller, Tünde Leviczky, Aladàr Pettkó‐Szandtner, Zsuzsanna Darula, Akitoshi Iwamoto, Mika Nomoto, Yasuomi Tada, Tetsuya Higashiyama, Taku Demura, John H. Doonan, Marie‐Theres Hauser, Keiko Sugimoto, Masaaki Umeda, Zoltán Magyar, László Bögre, Masaki Ito,
Tópico(s)Plant Reproductive Biology
ResumoArticle9 June 2015free access Transcriptional repression by MYB3R proteins regulates plant organ growth Kosuke Kobayashi Kosuke Kobayashi Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Toshiya Suzuki Toshiya Suzuki Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan JST, CREST, Chikusa, Nagoya, Japan Search for more papers by this author Eriko Iwata Eriko Iwata Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Norihito Nakamichi Norihito Nakamichi WPI Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya, Japan Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Takamasa Suzuki Takamasa Suzuki Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Poyu Chen Poyu Chen Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Misato Ohtani Misato Ohtani Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author Takashi Ishida Takashi Ishida Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan Search for more papers by this author Hanako Hosoya Hanako Hosoya Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan Search for more papers by this author Sabine Müller Sabine Müller Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany Search for more papers by this author Tünde Leviczky Tünde Leviczky Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Aladár Pettkó-Szandtner Aladár Pettkó-Szandtner Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Zsuzsanna Darula Zsuzsanna Darula Laboratory of Proteomic Research, Biological Research Centre, Szeged, Hungary Search for more papers by this author Akitoshi Iwamoto Akitoshi Iwamoto Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan Search for more papers by this author Mika Nomoto Mika Nomoto Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Yasuomi Tada Yasuomi Tada Center for Gene Research, Division of Biological Science, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Tetsuya Higashiyama Tetsuya Higashiyama WPI Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya, Japan Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Taku Demura Taku Demura Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author John H Doonan John H Doonan The National Plant Phenomics Centre, Aberystwyth University, Aberystwyth, UK Search for more papers by this author Marie-Theres Hauser Marie-Theres Hauser Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Keiko Sugimoto Keiko Sugimoto RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author Masaaki Umeda Masaaki Umeda Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan JST, CREST, Ikoma, Nara, Japan Search for more papers by this author Zoltán Magyar Zoltán Magyar Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK Search for more papers by this author László Bögre László Bögre Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK Search for more papers by this author Masaki Ito Corresponding Author Masaki Ito Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan JST, CREST, Chikusa, Nagoya, Japan Search for more papers by this author Kosuke Kobayashi Kosuke Kobayashi Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Toshiya Suzuki Toshiya Suzuki Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan JST, CREST, Chikusa, Nagoya, Japan Search for more papers by this author Eriko Iwata Eriko Iwata Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Norihito Nakamichi Norihito Nakamichi WPI Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya, Japan Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Takamasa Suzuki Takamasa Suzuki Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Poyu Chen Poyu Chen Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Misato Ohtani Misato Ohtani Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author Takashi Ishida Takashi Ishida Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan Search for more papers by this author Hanako Hosoya Hanako Hosoya Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan Search for more papers by this author Sabine Müller Sabine Müller Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany Search for more papers by this author Tünde Leviczky Tünde Leviczky Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Aladár Pettkó-Szandtner Aladár Pettkó-Szandtner Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Search for more papers by this author Zsuzsanna Darula Zsuzsanna Darula Laboratory of Proteomic Research, Biological Research Centre, Szeged, Hungary Search for more papers by this author Akitoshi Iwamoto Akitoshi Iwamoto Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan Search for more papers by this author Mika Nomoto Mika Nomoto Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Yasuomi Tada Yasuomi Tada Center for Gene Research, Division of Biological Science, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Tetsuya Higashiyama Tetsuya Higashiyama WPI Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya, Japan Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Chikusa, Nagoya, Japan Search for more papers by this author Taku Demura Taku Demura Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author John H Doonan John H Doonan The National Plant Phenomics Centre, Aberystwyth University, Aberystwyth, UK Search for more papers by this author Marie-Theres Hauser Marie-Theres Hauser Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria Search for more papers by this author Keiko Sugimoto Keiko Sugimoto RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Search for more papers by this author Masaaki Umeda Masaaki Umeda Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan JST, CREST, Ikoma, Nara, Japan Search for more papers by this author Zoltán Magyar Zoltán Magyar Institute of Plant Biology, Biological Research Centre, Szeged, Hungary Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK Search for more papers by this author László Bögre László Bögre Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK Search for more papers by this author Masaki Ito Corresponding Author Masaki Ito Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan JST, CREST, Chikusa, Nagoya, Japan Search for more papers by this author Author Information Kosuke Kobayashi1,‡, Toshiya Suzuki1,2,‡, Eriko Iwata1, Norihito Nakamichi3,4, Takamasa Suzuki4,5,18, Poyu Chen6, Misato Ohtani6,7, Takashi Ishida8, Hanako Hosoya9, Sabine Müller10, Tünde Leviczky11, Aladár Pettkó-Szandtner11, Zsuzsanna Darula12, Akitoshi Iwamoto9, Mika Nomoto4, Yasuomi Tada13, Tetsuya Higashiyama3,4,5, Taku Demura6,7, John H Doonan14, Marie-Theres Hauser15, Keiko Sugimoto7, Masaaki Umeda6,16, Zoltán Magyar11,17, László Bögre17 and Masaki Ito 1,2 1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan 2JST, CREST, Chikusa, Nagoya, Japan 3WPI Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya, Japan 4Graduate School of Sciences, Nagoya University, Chikusa, Nagoya, Japan 5JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Chikusa, Nagoya, Japan 6Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan 7RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan 8Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan 9Department of Biology, Tokyo Gakugei University, Koganei, Tokyo, Japan 10Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany 11Institute of Plant Biology, Biological Research Centre, Szeged, Hungary 12Laboratory of Proteomic Research, Biological Research Centre, Szeged, Hungary 13Center for Gene Research, Division of Biological Science, Nagoya University, Chikusa, Nagoya, Japan 14The National Plant Phenomics Centre, Aberystwyth University, Aberystwyth, UK 15Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria 16JST, CREST, Ikoma, Nara, Japan 17Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK 18Present address: College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 52 789 4168; Fax: +81 52 789 4165; E-mail: [email protected] The EMBO Journal (2015)34:1992-2007https://doi.org/10.15252/embj.201490899 See also: M Fischer & JA DeCaprio (August 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size. Synopsis Activator and DREAM-like repressor R1R2R3-Myb transcription factors act coordinately to regulate mitotic target genes and organ growth in Arabidopsis thaliana. R1R2R3-Mybs with repressor functions maintain G2/M-specific genes repressed in post-mitotic cells and restrict mitotic gene expression in proliferating cells. Transcriptional cell cycle regulation at G2/M impacts on plant organ growth. R1R2R3-Myb activators and repressors form distinct multi-protein complexes. Repressive R1R2R3-Mybs are similar to the DREAM/dREAM complexes in human and fly. Introduction During organ development, cell proliferation and differentiation are regulated in a temporally and spatially coordinated manner. In general, there is a gradual decrease in cell division activity as organogenesis proceeds, and most, if not all, cells eventually stop dividing and differentiate. The scheduled cessation of cell division is critical for the formation of organs with genetically defined sizes and morphologies (Conlon & Raff, 1999; Potter & Xu, 2001; De Vos et al, 2012; Hepworth & Lenhard, 2014). Organ initiation and growth in plants are largely post-embryonic that relies almost entirely on the activity of meristems, which contain dividing undifferentiated cells and are maintained throughout the lifetime of plants (Scheres, 2007). As cells exit the meristematic zone, cell proliferation ceases which relies on the negative regulation of cell cycle progression, but the mechanisms are not fully understood (Gutierrez, 2005; de Jager et al, 2005; Inzé & De Veylder, 2006; Komaki & Sugimoto, 2012). Leaf and sepal growth is determinate, and these organs represent the most studied models for the temporal and spatial regulation of cell proliferation. At initial stages of organ development, active cell division leads to a rapid increase in the number of cells within the primordia, which is followed by the gradual decrease in cell division activities (Beemster et al, 2005; Roeder et al, 2010; Andriankaja et al, 2012). In Arabidopsis, the cessation of cell division is associated with the onset of endoreduplication, in which DNA replication is repeated without intervening mitosis, leading to an increase in cellular DNA content (De Veylder et al, 2011; Fox & Duronio, 2013). As differentiation takes place, cells enter a quiescent state that is typically maintained for the rest of the plant's life. Similar temporal changes in cell division activity occur during the indeterminate growth of the root, generating an apical–basal positional gradient of cell division activity (Vanstraelen et al, 2009; Ivanov & Dubrovsky, 2013). In Arabidopsis, genome-wide expression profiling uncovered the dynamic transcriptional regulation during root and leaf development (Birnbaum et al, 2003; Beemster et al, 2005). The cluster of G2/M-phase-specific genes showed rapid and pronounced downregulation as cells differentiate which was correlated with the cessation of cell division. However, it is not clear whether such downregulation is an active process that is mediated by developmentally regulated transcriptional repression, or whether it is an indirect consequence of decreased cell proliferation activity. It is widely accepted that transcriptional regulations are essential for the developmental control of cell division (Berckmans & De Veylder, 2009). One of the important mechanisms for such regulation is based on the retinoblastoma (RB)-E2F pathway, which regulates the expression of many genes required for cell proliferation. The conserved tumor suppressor RB, called RB-related (RBR) in Arabidopsis, is known to associate with three functionally distinct E2F transcription factors. RBR may repress cell proliferation through E2FB (Magyar et al, 2012), while E2FC acts as transcriptional repressor and is required for the timed cessation of cell division and occurrence of endoreduplication during leaf development (del Pozo et al, 2006; de Jager et al, 2009). Thus, both RBR1 and E2FC act as negative regulators for cell division and are required for scheduled exit from the mitotic cell cycle that might be important to set up the developmental gradient of cell division activities in growing organs. In mammalian cells, DP1, the RB-related protein p130 and E2F4, together with the MuvB complex (containing LIN9, LIN37, LIN52, LIN54, and RBBP4), make a multiprotein complex known as DP, RB-like E2F, and MuvB (DREAM) complex (Litovchick et al, 2007; Sadasivam & DeCaprio, 2013). Current evidence suggests that this complex acts as a repressor of cell cycle-regulated genes during quiescence (Litovchick et al, 2007). When a quiescent mammalian cell is stimulated to enter the cell cycle, the complex releases p130, DP1, and E2F4 and instead recruits a member of the Myb transcription factors, B-MYB, to promoters of G2/M-specific genes. This recruitment is required for transcriptional activation of various genes essential for mitosis (Sadasivam et al, 2012). A similar multiprotein complex called Drosophila RBF, E2F2, and Myb (dREAM) is known in flies, which acts for repression of a variety of developmentally regulated genes and also for activation of the mitotic genes in proliferating cells (Korenjak et al, 2004; Georlette et al, 2007). The latter function of dREAM complex is attributed to dMYB, a single Myb gene in Drosophila (Beall et al, 2004; Lipsick, 2004). Accumulating evidence suggests that the conserved multiprotein complex has general roles as a global repressor and that Myb proteins counteract the repression (Sadasivam & DeCaprio, 2013). Although plants share the conserved members of E2F, DP, RB, Myb proteins (Vandepoele et al, 2002; Ito, 2005), and some MuvB components, the existence of DREAM/dREAM repressor complex and its possible functions are not known in plants. There are more than a hundred Myb genes encoded in plant genomes. However, most of Myb proteins in plants contain only two Myb repeats in N-terminal DNA-binding domain, as opposed to the three Myb repeats in animals (Dubos et al, 2010). The plant-specific two-repeat, R2R3-Myb genes have diverse roles in plant development and environmental responses. On the other hand, a small number of three Myb repeat-containing plant proteins, called R1R2R3-Myb or MYB3R, are linked to the regulation of mitosis (Ito, 2005). We have previously shown that plant MYB3R proteins regulate many G2/M-specific genes such as CYCB1, CYCB2, and CDKB2, by binding to the common cis-acting elements that are known as MSA element (Ito et al, 1998, 2001; Kato et al, 2009; Haga et al, 2011). In Arabidopsis, there are five genes that encode MYB3R transcription factors (Dubos et al, 2010). Our previous studies showed that two structurally related MYB3R proteins, MYB3R1 and MYB3R4, act as transcriptional activators on many, if not on all, G2/M-specific genes (Haga et al, 2011). We also showed that they are required for cytokinesis via the transcriptional activation of a critical target gene, KNOLLE (KN), a gene essential for cell plate formation (Lukowitz et al, 1996; Haga et al, 2007). Here, we report on the function of another pair of closely related MYB3R genes, MYB3R3 and MYB3R5, to be repressors of the transcription of G2/M-specific genes. The previously identified activator MYB3R1 can also have redundant repressor functions with MYB3R3 and MYB3R5 to inhibit the transcription of many G2/M-specific genes most pronouncedly in differentiated cells that have ceased to proliferate. The triple mutant of these three MYB3R genes shows hyperplasia, generating organs with increased sizes but also some developmental abnormalities and irregular cell divisions during embryogenesis. Genome-wide transcriptional profiling and chromatin immunoprecipitation experiments with MYB3R3 identified G2/M-specific target genes and show that MYB3R3 can also associate with promoters known to be E2F targets. However, the expression of these E2F target genes is not dependent on the repressor MYB3Rs. Accordingly, our biochemical data showed that MYB3R3 associates with E2FC and RBR1, while the activator MYB3R4 is found together with E2FB and RBR1. With mass spectrometry detection and pairwise interaction assays, we could also show other known DREAM/dREAM complex components together with MYB3R3, RBR1, and E2FB, but the exact composition of these complexes remains to be elucidated. We propose that the repressor MYB3R proteins may form complexes that are important for restricting the time window of mitotic gene expression in proliferating cells and for the maintenance of repressed states of G2/M-specific genes in post-mitotic cells. Results MYB3R1, MYB3R3, and MYB3R5 act redundantly as transcriptional repressors Phylogenetic analysis showed that there are two evolutionarily conserved groups in plant MYB3R family (Fig 1A). One contains MYB3R1 and MYB3R4 (hereafter MYB3R1/4) from Arabidopsis, which were previously shown to act as transcriptional activators (Ito et al, 2001; Haga et al, 2007, 2011). The other group contains two Arabidopsis MYB3Rs, MYB3R3 and MYB3R5 (hereafter MYB3R3/5), whose function was addressed in this study. We analyzed T-DNA insertion alleles of these genes, myb3r3-1, myb3r3-2, and myb3r5-1, all of which resulted in complete loss of normal transcripts (Fig 1B and Supplementary Fig S1). Both MYB3R3 alleles gave identical phenotypes when combined with the other myb3r mutants (see below), and thus are hereafter referred to as myb3r3. The myb3r1/4 double mutant was reported to have aberrant cytokinesis (Haga et al, 2007) suggesting that these two proteins positively regulate mitosis, a function which is not shared with MYB3R3/5, because combined triple and quadruple mutants do not influence these cytokinetic defects (Supplementary Fig S2). To analyze the roles for MYB3R3/5 in transcriptional regulation of G2/M-specific genes, we performed quantitative RT–PCR (qRT–PCR) analyses of seedlings with double myb3r3/5 mutation (Fig 1C, gray bars) and found significant upregulation of many, but not all of the G2/M-specific genes with MSA element, which include those encoding mitotic regulators, CYCB1;1, CYCB1;2, CDC20.1, and also microtubule-associated proteins with cytokinetic functions, PLEIADE (PLE)/MAP65-3 (Müller et al, 2004) and ENDOSPERM DEFECTIVE1 (EDE1) (Pignocchi et al, 2009). This indicated a repressor function for MYB3R3/5. Figure 1. Identification of R1R2R3-Myb proteins with a repressor function A. Phylogenetic analysis of R1R2R3-Myb proteins in plants. Protein names that begin with “Nt” are from tobacco, those that begin with “Os” are from rice, and MYB3R1–MYB3R5 are from Arabidopsis. Protein sequences within Myb domains were used to construct an unrooted phylogenetic tree. B. Schematic structures of the MYB3R3 and MYB3R5 genes. The insertion sites of the T-DNA in each mutant allele are indicated. Exons are indicated by boxes, where untranslated regions and protein coding regions are shown in white and black colors, respectively. C. Upregulation of G2/M-specific genes in the double myb3r3/5 and triple myb3r1/3/5 mutants. Transcript levels for a set of G2/M-specific genes were analyzed by qRT–PCR in wild-type (WT), myb3r3/5, and myb3r1/3/5 seedlings (10 DAG). Transcript level of histone H4 was also analyzed as a control. Expression levels of each transcript were normalized by the levels of ACT2 expression and are expressed as relative values with average levels of transcripts in all the plants analyzed being set to 1.0. Error bars represent standard deviation (SD) for n = 3. D, E. MYB3R1, MYB3R3, and MYB3R5 act redundantly in the repression of G2/M-expressed genes. A qRT–PCR analysis of EDE1 and CYCB1;1 showed that MYB3R1, but not MYB3R4, acts as a repressor that is redundant with MYB3R3 and MYB3R5 (D), and that MYB3R1, MYB3R3, and MYB3R5 act redundantly with different contributions for repression of the G2/M-specific genes (E). The qRT–PCR was performed using 10-day-old seedlings with the indicated genotypes, where plus indicates the wild-type form and minus indicates homozygous mutation for each MYB3R gene. Expression levels are expressed as relative values that were normalized to the levels of ACT2 expression. Error bars represent SD for n = 3. Download figure Download PowerPoint We then tested genetic interactions among MYB3Rs for regulating G2/M-specific genes, using qRT–PCR analysis. In the myb3r1/3/5 triple mutant, there is a further upregulation of G2/M-specific genes, EDE1 and CYCB1;1, compared to the double myb3r3/5 but not in the myb3r3/4/5 (Fig 1D). This raised the unexpected possibility that MYB3R1, but not MYB3R4, has redundant functions both with activator- and repressor-type MYB3Rs. In the myb3r1/3/5 triple mutant, a large cohort of G2/M-specific genes are further upregulated in comparison to the double myb3r3/5 as shown by qRT–PCR (Fig 1C, black bars). To gain insight whether this might also be the case on a genome-wide scale for all mitotic genes, we performed microarray expression profiling of seedlings (Supplementary Fig S3). Although this microarray analysis was done with a single biological replicate, the transcriptome data suggested that many genes annotated as “mitotic” or “G2/M-specific” were upregulated in myb3r1/3/5 seedlings as compared to wild type or the myb3r3/5 double mutant. It also suggested that this upregulation was specific for the gene sets annotated as “G2/M-specific” and “mitotic”, while genes related to other cell cycle phases were essentially unaffected (Supplementary Fig S4, list of each gene set is found in Supplementary Table S1). To further investigate the genetic interactions between activator- and repressor-type MYB3Rs on mitotic gene regulation, we analyzed mitotic gene expression profiles in myb3r mutant combinations (Supplementary Fig S5, see quantitative expression data of individual mitotic genes in Supplementary Table S2). The single myb3r1 mutant had little impact on gene expression and myb3r4 showed minor downregulation, but in the double mutant, the downregulation was enhanced for a cohort of mitotic genes (Supplementary Fig S5). The single myb3r3 and myb3r5 mutants also displayed minor effects, but the double myb3r3/5 mutants showed upregulation for a distinct set of mitotic genes (Supplementary Fig S3). The upregulation of these genes was enhanced by introducing myb3r1 but not myb3r4. In the quadruple myb3r1/3/4/5 mutant, the upregulation and downregulation of these two cohorts of mitotic genes were combined (Supplementary Fig S5). The qRT–PCR analysis of EDE1 and CYCB1;1 in mutant combinations of myb3r1, myb3r3, and myb3r5 also confirmed that MYB3R1 can play redundant roles with repressor-type MYB3Rs and showed these repressor-type MYB3Rs act redundantly and contribute differently to the transcriptional repression (Fig 1E). To validate the microarray results obtained from different mutant seedlings, we selected representative genes for up-, downregulated, and unchanged clusters and performed qRT–PCR analysis in biological triplicates (Supplementary Fig S6). This experiment fully confirmed the microarray data and our conclusion that MYB3R1 has redundant functions both with the activator MYB3R4 and the repressor MYB3R3/5. Hereafter, we refer to MYB3R1/3/5 as repressor MYBs (Rep-MYBs) and MYB3R1/4 as activator MYBs (Act-MYBs). Repression of G2/M-specific genes in post-mitotic cells To address genetically whether or not the Act-MYB and Rep-MYB act antagonistically in the same cells, we aimed to correlate the expression of a critical mitotic target gene, KN and the cytokinetic phenotypes in the quadruple myb3r1/3/4/5 mutant. We found that the expression of KN, which is known to be downregulated in myb3r1/4 (Haga et al, 2007), but increased in myb3r1/3/5, came close to wild-type levels in the quadruple myb3r1/3/4/5 mutant, but the cytokinetic defect remained (Fig 2A). This argues against that Act-MYB and Rep-MYB act in the same cell, but rather suggest that their functions are spatially and/or temporally separated. We reasoned that the loss of Rep-MYB might affect the expression of KN and other G2/M-specific genes in post-mitotic cells, which would not lead to the rescue of cytokinetic defects caused by the loss of Act-MYB in dividing cells. Figure 2. MYB3R1, MYB3R3, and MYB3R5 act as repressors in post-mitotic cells during organ development Genetic interactions between repressor and activator MYBs. The frequencies of cytokinesis-defective stomata (n = 6) and levels of KN transcripts (n = 3) were quantified using seedlings (9 DAG) with mutations in either repressor MYBs (ΔRep), activator MYBs (ΔAct), or both (ΔActRep). The qRT–PCR data were normalized by the levels of ACT2 expression. Error bars represent SD. Expression of G2/M-specific genes during leaf development. The first leaf pairs were harvested from wild-type, myb3r3/5, and myb3r1/3/5 plants at indicated times after germination and
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