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First published online 7 February 2007
doi: 10.1242/dev.02801

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R1R2R3-Myb proteins positively regulate cytokinesis through activation of KNOLLE transcription in Arabidopsis thaliana

¹ Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
² Central Research Institute, Ishihara Sangyo Kaisha, Ltd, 2-3-1 Nishi-shibukawa, Kusatsu, Shiga 525-0025, Japan.
³ RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan.
⁴ Molecular and Cellular Breeding Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Sciences and Technology (AIST), Tsukuba Central 6, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
⁵ Zentrum für Molekularbiologie der Pflanzen (ZMBP), Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Molecular characterization of MYB3R1 and MYB3R4. (A) Phylogenetic relationship of R1R2R3-Myb proteins in plants. The phylogenetic tree was constructed based on the amino acid sequence similarities in the Myb domains of R1R2R3-Myb proteins from tobacco (NtmybA1, NtmybA2 and NtmybB), Arabidopsis (MYB3R1-MYB3R5) and rice (Os01g12860, Os12g13570, Os01g62410 and Os05g38460). (B) Protein structure of MYB3R1 and MYB3R4. The Myb domain is located in the N-terminus and contains tandemly repeated sequences that are designated R1, R2, and R3 (black boxes). Two separate regions in the C-terminal half show sequence similarity to their homologs in tobacco and rice (hatched boxes). (C) Position of T-DNA insertions in MYB3R1 and MYB3R4. Boxes and lines represent exons and introns, respectively. Arrows above the boxes indicate positions of T-DNA insertions. Black and white boxes correspond to coding and non-coding regions, respectively. Arrowheads indicate the positions of primers used for quantitative RT-PCR. (D) Transcript levels of MYB3R1 and MYB3R4 in wild-type and T-DNA insertion mutants. Levels of each transcript were determined by real-time quantitative RT-PCR using the primer pairs shown in C.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Defective cytokinesis in myb3r1 myb3r4 plants. (A) Wild-type proembryos at the two-cell stage. (B) Rod-shaped myb3r1 myb3r4 embryo with multiple nuclei that were not separated by internal cell walls. (C) myb3r1 myb3r4 embryo without internal cross walls, containing multiple metaphase chromosomes (arrowheads). (D) Normally developed stoma in wild-type silique valve. (E-G) Cytokinesis-defective single-celled stomata in myb3r1 myb3r4 silique valves. (E) Stoma consists of single guard cell with two internal cell wall stubs, lacking a detectable pore. (F) Stoma with incomplete internal cell wall that forms a small pore. (G) Stoma without internal pore-forming cell wall. (H,I) DAPI-stained stomatal nuclei in wild-type (J) and myb3r1 myb3r4 (K) silique valve. Note two nuclei in single-celled stoma of myb3r1 myb3r4. Red, autofluorescence of chlorophyll. (J-M) Frequent occurrence of cytokinesis-defective cells with gapped cell walls and cell wall stubs (asterisks) in various organs of myb3r1 myb3r4 plants. (J) Toluidine Blue-stained outer epidermis of silique valve. (K) Outer integument of ovule. (L) Epidermis of filament in stamen. (M) A longitudinally elongated layer of chlorenchyma in valve of silique. (N) Frequency of cytokinesis-defective stomata in various organs of myb3r1 myb3r4 plants, as determined by counting normal and defective stomata in the epidermis of each organ. A-G,K-M are DIC images. Scale bars: 10 µm in A-D,H; 20 µm in J-M.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Mutations in MYB3R1 and MYB3R4 genes affect the abundance of G2/M phase-specific transcripts. (A) Relative transcript levels of genes associated with cell-cycle regulation and cytokinesis in the myb3r1 myb3r4 plants. G2/M phase-specific genes that contain MSA elements are indicated by the black bar. (B) Relative transcript levels of KN and CYCB2;1 genes in plants with various allele combinations of MYB3R1 and MYB3R4: +/+, homozygous for wild-type allele; +/-, heterozygous; -/-, homozygous for mutant allele. All measurements in A and B were performed on inflorescences containing young flower buds. Expression levels were determined by real-time quantitative RT-PCR and normalized relative to those in wild-type plants. Columns represent mean values; error bars represent s.d.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Genetic interaction between myb3r1, myb3r4 and kn. (A) Effects of heterozygous mutation of kn on the severity of cytokinesis defects. Frequency of cytokinesis-defective stomata and epidermal cells were determined in the silique valve of the plants with the indicated genotypes. Data for myb3r1/myb3r1 myb3r4/myb3r4 KN/kn plants are not available because these plants do not produce siliques. Columns represent mean values; error bars represent s.d. (B-D) Defective cytokinesis in myb3r1/myb3r1 myb3r4/myb3r4 KN/kn plants. (B) Pavement epidermal cells and (C) palisade cells in rosette leaves, showing cell wall stubs (arrowheads). (D) Enlarged cells (arrowhead) with multiple nuclei (asterisks) in root epidermal cells. Scale bars in B-D, 20 µm. (E) Transcript levels of KN in plants with various allele combinations of MYB3R1, MYB3R4 and KN genes. All measurements were performed on inflorescences containing young flower buds in mature plants. Expression levels were determined by real-time quantitative RT-PCR and normalized relative to those in wild-type plants. Columns represent mean values; error bars represent s.d.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Two MSA elements are required for sufficient expression of the KN gene. (A) The KN gene contains three MSA motifs in different orientations in its promoter region (arrowheads). The promoter-deletion construct KNMSA1 only contains a promoter fragment from -286 bp to the KN start codon (ATG, +1). In the KNMSA2 construct, two MSA motifs and the sequence between them (-278 to -250 bp) were replaced by a 12-bp sequence containing XbaI and SmaI restriction sites. (B) Western blots of Myc-tagged KN from transgenic kn mutant plants carrying KNMSA1. (C) Western blots of Myc-tagged KN from transgenic plants carrying Myc-KN rescue construct and KNMSA2. In all three independent KNMSA2 transgenic plants, only endogenous KN without the Myc-tag was detected. KN, anti-KN antiserum; Myc, peroxidase-coupled anti-Myc monoclonal antibody.

View larger version (16K): [in this window] [in a new window]   Fig. 6. MYB3R1 and MYB3R4 act as transcriptional activators. (A) Activation of KN gene promoter by MYB3R1 and MYB3R4. KN::LUC reporter plasmid and 35S::MYB3R1 or 35S::MYB3R4 expression plasmid were co-transfected into tobacco BY-2 protoplasts, either with or without 35S::CYCB1. (B) MSA elements are required for MYB3R4-induced activation of the KN promoter. The KNMSA::LUC construct contains the KN promoter in which all three MSA elements are mutated. KN::LUC and KNMSA::LUC were transfected into tobacco BY-2 protoplasts together with 35S::MYB3R4 expression plasmids, either with or without 35S::CYCB1. (C) Effects of MYB3R4 on the activity of G2/M phase-specific promoters. Each promoter was fused to LUC and transfected into tobacco BY-2 protoplasts together with 35S::MYB3R4 expression plasmids, either with or without 35S::CYCB1. All LUC activities in A, B and C are normalized relative to the control transfection without expression plasmid. Bars represent mean values in each of five independent transfections; error bars represent s.d.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Expression of MYB3R1 and MYB3R4. (A,B) Expression during the cell cycle. (A) Change in transcript abundance during the cell cycle in synchronized MM2d cells. Transcript levels of each gene were analyzed by quantitative RT-PCR. (B) Change in mitotic index of the synchronized MM2d cells. (C-L) Expression domains of MYB3R1::GUS (C,E,G,I,K) and MYB3R4::GUS fusions (D,F,H,J,L). (C,D) 12-day-old seedling. (E,F) Emerging lateral roots. (G,H) Root tips. (I,J) Flowers. (K,L) Ovules with developing embryos. Scale bars: 50 µm.