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, 22 (11), 3778-90

The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis Thaliana

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The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis Thaliana

Ken Kosetsu et al. Plant Cell.

Abstract

Cytokinesis in plants is achieved by the formation of the cell plate. A pathway that includes mitogen-activated protein (MAP) kinase kinase kinase and MAP kinase kinase (MAPKK) plays a key role in the control of plant cytokinesis. We show here that a MAP kinase, MPK4, is required for the formation of the cell plate in Arabidopsis thaliana. Single mutations in MPK4 caused dwarfism and characteristic defects in cytokinesis, such as immature cell plates, which became much more prominent upon introduction of a mutation in MKK6/ANQ, the MAPKK for cytokinesis, into mpk4. MKK6/ANQ strongly activated MPK4 in protoplasts, and kinase activity of MPK4 was detected in wild-type tissues that contained dividing cells but not in mkk6/anq mutants. Fluorescent protein-fused MPK4 localized to the expanding cell plates in cells of root tips. Expansion of the cell plates in mpk4 root tips appeared to be retarded. The level of MPK11 transcripts was markedly elevated in mpk4 plants, and defects in the mpk4 mpk11 double mutant with respect to growth and cytokinesis were more severe than in the corresponding single mutants. These results indicate that MPK4 is the downstream target of MKK6/ANQ in the regulation of cytokinesis in Arabidopsis and that MPK11 is also involved in cytokinesis.

Figures

Figure 1.
Figure 1.
Effects of mpk4 Alleles on the Growth and Organ Morphology of Arabidopsis and on Cell Division in Two Different Ecotypes, Col-0 and Ler. (A) to (D) Gross morphology of wild-type (Wt; Col-0; [A]), mpk4-1 (Ler; [B]), mpk4-2 (Col-0; [C]), and mpk4-1 (Col-0; [D]) plants (12-d-old plants). Bars = 1 cm. (E) Sites of Ds transposon and T-DNA insertions in the alleles. (F) Analysis of transcripts. RT-PCR was performed with total RNA prepared from wild-type (Col-0; lane 1), mpk4-1 (lane 2), and mpk4-2 (lane 3) plants with primer pairs specific for the indicated genes. The number of cycles of PCR is indicated at the right of each panel. (G) to (J) Dark-field views of roots of wild-type (Col-0; [G]), mpk4-1 (Ler; [H]), mpk4-2 (Col-0; [I]), and mpk4-1 (Col-0; [J]) plants. Bars = 0.25 mm. (K) to (V) Transverse sections of roots ([K] to [N]) and cotyledons ([O] to [V]) of 14-d-old plants stained with toluidine blue. Sections of the roots of wild-type (Col-0; [K]), mpk4-1 (Ler; [L]), mpk4-2 (Col-0; [M]), and mpk4-1 (Col-0; [N]) plants. Bars = 50 μm. Transverse sections of the cotyledons of wild-type (Col-0; [O]), mpk4-1 (Ler; [Q]), mpk4-2 (Col-0; [S]), and mpk4-1 (Col-0; [U]) plants. Magnified views of boxed regions are shown in (P), (R), (T), and (V). Arrowheads and arrows in panels indicate nuclei in multinucleate cells and incomplete cross walls, respectively. Bars = 100 μm in (O), (Q), (S), and (U) and 50 μm in (P), (R), (T), and (V).
Figure 2.
Figure 2.
Comparison of the Phenotypes of the mpk4-2 mkk6-2/anq-2 and mpk4-2 mpk11 Double Mutants with That of Each Respective Single Mutant. (A) to (D) Gross morphology of wild-type (Wt; Col-0) (A), mpk4-2 (B), mkk6-2/anq-2 (C), and mpk4-2 mkk6-2/anq-2 (D) plants (15-d-old plants). All plants shown here had the Col-0 background. Plants were grown on MS basic medium. Bars = 0.5 cm. (E) Phylogenetic tree for tobacco NRK1 and five MPKs in the group B subfamily. Amino acid sequences of tobacco NRK1 and group B subfamily MAPKs were aligned with the ClustalX program, and the resulting alignment was used to generate a phylogenetic tree with GENETYX-Mac version 13. Values indicate the number of times that each branch topology was found during bootstrap analysis. (F) Accumulation of transcripts of MKK6/ANQ MAPKK and five genes for MPKs in the indicated organs of the wild-type plant, as determined by real-time RT-PCR with primers specific for each respective gene. Quantitative RT-PCR data were normalized by reference to expression of the gene for β-tubulin, and values relative to that for mature leaves are shown. Error bars indicate sd (n = 3). (G) to (J) Gross morphology of wild-type (G), mpk11 (H), mpk4-2 (I), and mpk4-2 mpk11 (J) plants (17-d-old plants). All plants shown here had the Col-0 background. Plants were grown on MS basic medium. Bars = 1 cm.
Figure 3.
Figure 3.
MPK4 Represses the Accumulation of MPK11 Transcripts. (A) and (B) Quantitative RT-PCR was performed to determine the levels of transcripts of MPK4, MPK5, MPK11, MPK12, and MPK13 in wild-type (Col-0), mpk4-2, mkk6-2/anq-2, and nack1-3 plants. All plants analyzed here had the Col-0 background. Total RNA was prepared from the shoot apex (A) and cotyledons (B) of 9-d-old plants. After the reverse transcription reaction, the resultant cDNAs were amplified with primers specific for each respective gene. Quantitative RT-PCR data were normalized by reference to transcripts of the EF1-α gene. Values are shown relative to that for the wild type. Error bars indicate sd (n = 3).
Figure 4.
Figure 4.
Identification of Candidate MAPKs (MPK4, MPK5, MPK11, MPK12, and MPK13) Downstream of MKK6/ANQ MAPKK. DNA constructs for the various HA-tagged MAPKs (MAPK-HA) were transiently introduced into protoplasts of Arabidopsis. Activation of MAPKs was examined by coexpression of Myc-tagged MKK6/ANQ (MKK6/ANQ-Myc) with a kinase-inactive domain (L), the wild-type domain (W), or a constitutively active domain (G). The kinase activity of MAPK-HA that had been immunoprecipitated with HA-specific antibodies was assayed with MBP as substrate, and [γ-32P]ATP phosphorylation of MBP was quantitated by autoradiography after SDS-PAGE. MAPK-HA and MKK6/ANQ-Myc proteins were detected by protein gel blotting with HA-specific (for MAPKs) and Myc-specific (for MKK6/ANQ) antibodies, respectively.
Figure 5.
Figure 5.
Elevated Activity of MPK4 Protein Kinase in Organs and Tissues That Contain Dividing Cells. (A) Endogenous activities of MPK4 in various organs. Protein extracts were obtained from the indicated organs and tissues of Col-0 wild-type (Wt) and mpk4-2 9-d-old plants. Immunocomplex kinase assays were performed with MPK4-specific antibodies and MBP as substrate. Whole wild-type (1) and mpk4-2 (2) plants, and the developing first and second leaves (3), cotyledons (4), shoot apices (5), roots without tips (6), and root tips (7) of wild-type plants were examined (top). MBP was visualized after staining with Coomassie blue (middle). Endogenous levels of MPK4 protein in the various organs are shown in the bottom panel. Extracts used for immunocomplex kinase assays were immunoblotted with MPK4-specific antibodies. Twenty micrograms of protein were loaded in each lane. (B) Endogenous activity of MPK4 protein kinase in the wild-type (Col-0), mpk4-2, and mkk6-2/anq-2 plants. Immunocomplex kinase assays were performed with MPK4-specific antibodies as described above (top). The protein extracts were obtained from developing first and second leaves plus shoot apices of wild-type, mpk4-2, and mkk6-2/anq-2 plants. MBP was visualized after staining with Coomassie blue (middle). Levels of MPK4 proteins are shown in the bottom panel. Extracts used for immunocomplex kinase assays were immunoblotted with MPK4-specific antibodies. Twenty micrograms of protein were loaded in each lane. The signal detected in the case of mpk4-2 was due to nonspecific MAPK.
Figure 6.
Figure 6.
MPK4 Is Localized to Cell Plates and Involved in Its Expansion in Cells in the Division Zone of Roots. (A) and (B) The photographs, taken under a confocal microscope, show the fluorescence due to YFP in the root tip of a transgenic plant that expressed the fusion gene composed of the entire MPK4 locus and YFP cDNA. (C) to (E) Magnified views of boxed regions that are shown in (A) and (B). Arrows and arrowheads indicate regions that appeared to correspond to the division plane of the cells and the two newly formed nuclei, respectively. (F) to (H) Fluorescence micrographs show the fluorescence due to FM4-64 (F) and MPK4-GFP (G) in the root tip of a transgenic plant that expressed the fusion gene composed of the entire MPK4 locus and GFP cDNA. Merged images are shown in (H). Bars = 10 μm. (I) to (K) Time series of growing cell plates stained with FM4-64 in root tips of 3-d-old wild-type (I) and mpk4-2 ([J] and [K]) plants. Arrows indicate the growing or arrested cell plates. Cells in these pictures are located 70 to 90 μm above quiescent center. Bars = 5 μm.
Figure 7.
Figure 7.
Hypothetical Scheme Showing the Possible Signaling Circuitry That Controls Cytokinesis in Arabidopsis. Components in the NACK-PQR pathway are shown. The MPK4 MAPK plays a crucial role downstream of MKK6/ANQ MAPKK during formation of the cell plate. MPK4 might repress transcription of the MPK11 gene in a cytokinesis-independent manner (1); alternatively, the proper progression of the formation of the cell plate that is mediated by MPK4 might repress the transcription of MPK11 (cytokinesis-dependent mechanism) (2).

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