Transcriptional repression by MYB3R proteins regulates plant organ growth

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.

Keywords: DREAM complex; G2/M phase; MYB transcription factors; cell cycle regulation; cell differentiation.

Figure 1

Identification of R1R2R3-Myb proteins with a repressor function

1. 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.

2. 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.

3. 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.

4. 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.

Figure 2

MYB3R1, MYB3R3, and MYB3R5 act as repressors in post-mitotic cells during organ development

1. 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.

2. 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 were used for qRT–PCR analysis to determine the transcript levels of EDE1 and KNOLLE. CYCD3;1 was also analyzed as a control with an expression that is dependent on cell division, but not directly regulated by MYB3Rs. Images of representative wild-type plants at indicated times are also shown, in which the first leaf pairs are indicated by arrowheads.

3. Preferential upregulation of G2/M-specific genes in old leaves upon loss of repressor MYBs. Microarray analysis of the first leaf pair was conducted in wild-type and myb3r1/3/5 plants, and the expression signals are shown as scattered plots where red dots indicate G2/M-specific genes.

4. Upregulation of G2/M-specific genes in various organs upon loss of repressor MYBs. Various organs at different developmental stages were harvested and used for qRT–PCR analysis to determine the expression levels of CYCB1;2 and EDE1. The qPCR data were normalized by the levels of ACT2 expression. Data are shown as fold upregulation in myb3r1/3/5 compared to wild-type plants. Error bars represent SD for n = 3.

Data Information: WT, wild type; Δ3,5, myb3r3/5 double mutant; Δ1,3,5, myb3r1/3/5 triple mutant.

Figure 3

Ectopic expression of G2/M-specific genes in proliferating and post-mitotic quiescent cells upon loss of repressor MYBs

1. Expression of CYCB1;2-YFP in the leaf epidermis of 9-day-old plants. Leaves from myb3r1/3/5 (Δ1,3,5) and wild-type (WT) plants were counterstained with propidium iodide for cell walls to visualize cell outlines and were analyzed by confocal microscopy. In myb3r1/3/5 leaves, CYCB1;2-YFP expression was often observed in cells with enlarged nuclei that had presumably undergone endoreduplication, as indicated by asterisks.

2. Expression of CYCB1;2-YFP in roots of 3-day-old seedlings. CYCB1;2-YFP expression was expanded toward the basal zone of roots in myb3r1/3/5 seedlings.

3. Ectopic expression of CYCB1;2-YFP in terminally differentiated root hair cells in myb3r1/3/5 seedlings (asterisks).

4. Expression of proAtNACK1::YFP in epidermal non-dividing cells in myb3r1/3/5 hypocotyl (asterisks).

5. Ectopic expression of proEDE1::YFP in maturing guard cells in myb3r1/3/5 leaves (asterisks). Such expression was absent in wild-type leaves (arrowheads).

6. Expression of CYCB1;2-YFP in the developing embryo. In a myb3r1/3/5 embryo, a greater population of cells expressed CYCB1;2-YFP compared with a wild-type embryo.

7. Expression of CYCB1;2-YFP in cotyledon from 3-day-old seedlings. Asterisks indicate YFP expression in endoreduplicated cells with enlarged nuclei.

8. Quantitative comparison of CYCB1;2-YFP-expressing cells in myb3r1/3/5 and wild-type plants. Proportion of CYCB1;2-YFP-expressing cells was determined among epidermal cells in root tips of 5-day-old seedlings (n = 12), and meristemoids and guard mother cells (GMC) in first leaf pairs of 9-day-old seedlings (n = 8). Error bars represent SD. The asterisks in the graphs show differences that are statistically significant (t-test P-value < 0.05).

Data Information: WT, wild type; Δ1,3,5, myb3r1/3/5 triple mutant. Scale bars, 50 μm in (A–E, G), 200 μm in (F).

Figure 4

Genetic interactions between myb3r1/3/5 and ple-2 or ede1-1 reveal the functions of repressor MYBs in proliferating cells

1. Cytokinesis defects in ple-2 roots were partially suppressed by combinational mutations in MYB3R1/3/5. The roots of plants with the indicated genotypes were stained at 7 DAG by propidium iodide to visualize cell outlines and were analyzed by confocal microscopy. Magnified views of the boxed area are provided below each image, to show cytokinesis defects, such as gapped cell walls and cell wall stubs (indicated by asterisks).

2. Cytokinesis defects in ede1-1 cotyledons were partially suppressed by combinational mutations in MYB3R1/3/5. Cotyledons of plants (8 DAG) with the indicated genotypes were fixed, cleared, and observed by DIC microscopy. The ede1-1 mutation causes incomplete cytokinesis of guard mother cells, producing single-celled stomata (asterisk).

Data Information: WT, wild type; Δ3,5, myb3r3/5 double mutant; Δ1,3,5, myb3r1/3/5 triple mutant. Scale bars, 50 μm in (A), 20 μm in (B).

Figure 5

Genome-wide identification of MYB3R3-bound genes in vivo

1. MYB3R3-GFP associates with promoters of G2/M-specific genes. ChIP–qPCR assays were performed using whole seedlings of MYB3R3-GFP, and the results are shown as the percentage of DNA fragments co-immunoprecipitated with anti-GFP antibody relative to input DNA. Grey and black bars indicate the results from MYB3R3-GFP and wild-type seedlings, respectively. Each measurement was performed twice and produced similar results.

2. Both G2/M-specific genes and E2F target genes are enriched in MYB3R3-bound chromatin regions. Venn diagram analysis was conducted to compare MYB3R3-bound genes with the indicated gene categories. There are significant overlaps between MYB3R3-bound and G2/M-specific genes, and between MYB3R3-bound and E2F target genes with P-value < 10⁻¹⁵ in Fisher’s exact test. Numbers represent the gene number in each category.

3. Enrichment of the MSA and E2F motifs in MYB3R3-bound chromatin regions. Enriched sequences in immunoprecipitated DNA faction were searched using motif-finding software MEME-ChIP web tool (http://meme.nbcr.net/meme/tools/meme-chip).

4. Peak distributions around CYCB1;2 and ORC1 genes in the ChIP-seq analysis of MYB3R3-GFP plants. CYCB1;2 and ORC1 genes are shown as the representative examples of G2/M-specific and E2F target genes, respectively.

Figure 6

MYB3R3 and MYB3R4 both interact with RBR1 and differently associate with E2F isoforms

1. A, B MYB3R3-GFP and GFP-MYB3R4 both interact with RBR1 and CDKA;1, but with a different E2F isoform in Arabidopsis leaves. IP was performed with anti-GFP antibodies from protein extracts prepared from first leaf pairs of MYB3R3-GFP or GFP-MYB3R4 transgenic plants at indicated days after germination (DAG). In these transgenic plants, expression of GFP fusion proteins was driven by the corresponding native promoters. Co-IP of RBR1 and E2FB was examined by Western blot analyses using corresponding antibodies. For detection of MYB3R3-GFP and CDKA;1, anti-GFP and anti-PSTAIRE (specific to CDKA;1) antibodies were used. As input, 1/10 of IP was loaded. Coomassie staining of the same membrane was used as a loading control.

2. C MYB3R3-GFP interacts with E2FC, but GFP-MYB3R4 does not. IP was performed with anti-GFP antibodies from protein extracts prepared from first leaf pairs of MYB3R3-GFP or GFP-MYB3R4 transgenic plants at indicated days after germination (DAG). Co-IP of E2FC and CDKA;1 was examined by Western blot analyses using anti-E2FC and anti-PSTAIRE antibodies, respectively. As input, 1/16 of IP was loaded. Coomassie staining of the same membrane was used as a loading control.

Figure 7

Loss of repressor MYBs causes enhanced organ growth and ectopic cell division

1. Increased seed size of myb3r1/3/5 triple mutant. The average seed area after imbibition was determined (n = 25).

2. Enlarged embryo of a myb3r1/3/5 triple mutant.

3. Enhanced leaf growth at the initial stage of leaf development in myb3r1/3/5 triple mutant. SEM images of initiating first leaf pairs at 4 and 5 DAG are shown. A graph shows the average length of initiating leaves of wild-type and myb3r1/3/5 seedlings (n = 6).

4. Increased cell number causes greater leaf size in myb3r1/3/5 triple mutants at the early seedling stage. Images show whole seedlings of wild-type and myb3r1/3/5 plants at 7 and 9 DAG. First leaf pairs are indicated by arrowheads. Graphs show that, at 7 DAG, the difference in the number of palisade cells per leaf, but not their size, is statistically significant (n = 5).

5. Time-course changes of ploidy levels in first leaf pairs of wild-type and myb3r1/3/5 seedlings. The levels of ploidy are expressed by the value called “endoreduplication index,” which represents the average number of endoreduplication cycles that were experienced by the cells (n = 5).

6. Ploidy distribution of first leaf pairs at 15 DAG. Representative results of flow cytometric analysis (left) and ploidy distributions (right) of wild-type and myb3r1/3/5 plants are shown (n = 5).

7. Root phenotypes in wild-type and myb3r1/3/5 plants. Images show seedlings at 6 DAG.

8. Increase in meristem size in myb3r1/3/5 roots. Images show propidium-iodide-stained roots at 5 DAG. Arrowheads indicate the basal end of the root meristem, which was determined by the position at which the cells start elongating.

9. Ectopic cell division during myb3r1/3/5 embryo development. A cell clump was formed in suspensor of myb3r1/3/5 embryo (asterisk).

10. An ectopic shoot apical meristem generated in a myb3r1/3/5 seedling.

Data Information: WT, wild type; Δ1,3,5, myb3r1/3/5 triple mutant. Scale bars: 500 μm in (A) and (B), and 100 μm in (C), (H), and (I). In all panels, error bars represent SD. The asterisks in the graphs show differences that are statistically significant (t-test P-value < 0.05).