Arabidopsis thaliana MAP65-1 and MAP65-2 function redundantly with MAP65-3/PLEIADE in cytokinesis downstream of MPK4

Abstract

Plant cytokinesis occurs by the growth of cell plates from the interior to the periphery of the cell. These dynamic events in cytokinesis are mediated by a plant-specific microtubule (MT) array called the phragmoplast, which consists of bundled antiparallel MTs between the two daughter nuclei. The NACK-PQR pathway, a NACK1 kinesin-like protein and mitogen activated protein kinase (MAPK) cascade, is a key regulator of plant cytokinesis through the regulation of phragmoplast MTs. The MT-associated protein MAP65 has been identified as one of the structural components of MT assays involved in cell division, and we recently showed that Arabidopsis AtMAP65-3/PLEIADE (PLE) is a substrate of MPK4 that is a component of the NACK-PQR pathway in Arabidopsis. Here we show that AtMAP65-1 and AtMAP65-2 are also phosphorylated by MPK4. AtMAP65-1 and AtMAP65-2 that localize to the phragmoplast were phosphorylated by MPK4 in vitro. Although mutants of the Arabidopsis AtMAP65-1 and AtMAP65-2 genes exhibited a wild-type phenotype, double mutations of AtMAP65-3 and AtMAP65-1 or AtMAP65-2 caused more severe growth and cytokinetic defects than the single atmap65-3/ple mutation. These results suggest that AtMAP65-1 and AtMAP65-2 also function in cytokinesis downstream of MPK4.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, and are involved in various signaling processes in plant development, cell division and responses to endogenous or exogenous stimuli.¹ The NACK-PQR pathway, one of the best-characterized MAPK cascades in plants, has been identified as a key regulator of plant cytokinesis in tobacco. This pathway is composed of NPK1 MAPK kinase kinase (MAPKKK), NQK1/NtMEK1 MAPK kinase (MAPKK), NRK1/NTF6 MAPK and NACK1 kinesin-like protein, an activator of NPK1 MAPKKK.^2–5 During cytokinesis, all these components are localized on the equator of the phragmoplast, which is the plant-specific cytokinetic apparatus organized by microtubules (MTs). Downstream of this pathway, tobacco MAP65-1, an MT-associated protein, is phosphorylated by NRK1/NTF6 MAPK and phosphorylated MAP65-1 is localized to the equator of the phragmoplast.⁶ Phosphorylation of MAP65-1 by NRK1/NTF6 decreases the ability of MAP65-1 to bundle MTs, suggesting that the NACK-PQR pathway regulates expansion of the phragmoplast through the phosphorylation of MAP65.⁶

The NACK-PQR pathway also seems to be conserved in Arabidopsis and rice. Several orthologs of components of the NACK-PQR pathway except for MAPK have been identified independently as regulators of cytokinesis in these plants.^3,5,7–14 Recently we reported that ANP MAPKKKs, MPK6/ANQ MAPKK and MPK4 MAPK biochemically constitute the MAPK pathway and HINKEL/AtNACK1 functions as an activator of ANP MAPKKKs.¹⁵ In addition, we revealed that MPK4 MAPK is localized to cell plates during cytokinesis, is required for cytokinesis in Arabidopsis and phosphorylates AtMAP65-3.¹⁶ Although AtMAP65-3 is proposed to be involved in cytokinesis,^17,18 and AtMAP65-1 is supposed to be a substrate of MPK4 based on a series of experiments,^6,19,20 the involvement in cytokinesis of other closely related members of the Arabidopsis MAP65 family, AtMAP65-1 and AtNAP65-2, has yet to be tested. In this report, we suggest redundant functions of these MAP65 molecules in cytokinesis of Arabidopsis.

AtMAP65-1 and AtMAP65-2 are Localized to the Phragmoplast and Phosphorylated by MPK4 in vitro

We tested whether AtMAP65-1 and AtMAP65-2 can be phosphorylated by MPK4 in vitro. We prepared histidine-tagged AtMAP65-1 (His-MAP65-1) and AtMAP65-2 (His-MAP65-2) proteins and performed kinase assays in vitro with activated recombinant MPK4. As shown in Figure 1A, His-MAP65-1 and His-MAP65-2 proteins were phosphorylated efficiently by MPK4 in the presence of GST-MKK6/ANQ, indicating that these proteins are also substrates of MPK4 in vitro (Fig. 1A).

Figure 1.

Phosphorylation in vitro of AtMAP65-1 and AtMAP65-2 by MPK4 MAPK. (A) Phosphorylation in vitro of AtMAP65-1 and AtMAP65-2 by MPK4. Recombinant His-MAP65-1 and His-MAP65-2 proteins were used as substrates in a kinase assay with His-T7-MPK4 or His-T7-MPK4 activated by GST-MKK6 in the presence of [γ-³²P]ATP. Kinase activity was assayed as previously reported in reference ¹⁶. MPK4-phosphorylated AtMAP65-1 and AtMAP65-2 proteins were detected by autoradiography (top). The SDS-PAGE gel was stained with Coomassie Brilliant Blue (CBB; bottom). (B) Subcellular localization of GFP-MAP65-1, GFP-MAP65-2 and GFP-MAP65-3 in BY-2 cells. cDNA constructs for GFP-MAP65s were controlled under the Dex-inducible promoter. BY-2 cells expressing GFP-MAP65-1 (a), GFP-MAP65-2 (b) and GFP-MAP65-3 (c) were incubated for 12 h in the presence of 0.5 µM Dex. Photographs, taken under a confocal microscope, show the fluorescence due to GFP in BY-2 cells during cytokinesis (left parts). Nomarski images (DIC ) and merged images are shown in center and right parts, respectively. Scale bars: 10 µm.

To examine the subcellular localization of AtMAP65-1 and AtMAP65-2, we generated DNA constructs that encoded green fluorescent protein (GFP) fused to AtMAP65-1 (GFP-MAP65-1) and AtMAP65-2 (GFP-MAP65-2) under the control of a dexamethasone (Dex)-inducible transcription system,²¹ and introduced them to cultured tobacco BY-2 cells. As shown in Figure 1B, both GFP-MAP65-1 and GFP-MAP65-2 were localized entirely to the phragmoplast during cytokinesis, whereas GFP-MAP65-3 was localized at the equatorial zone of the phragmoplast, as reported previously in references 16, 17, 22–26 (Fig. 1B). GFP-MAP65-2 was slightly concentrated at the midline of the phragmoplast (Fig. 1B(b)). Observations by confocal microscopy showed that GFP fluorescence derived from GFP-MAP65-1, GFP-MAP65-2 and GFP-MAP65-3 (Fig. 1B(c)) had ring-like profiles (data not shown), at least partially consistent with localization of MPK4 and MKK6/ANQ proteins.^15,16 These data suggest that AtMAP65-1, AtMAP65-2 and AtMAP65-3 redundantly regulate MT dynamics during cytokinesis through phosphorylation by MPK4 activated by MKK6/ANQ.

The Expression Levels of AtMAP65-2 and AtMAP65-3 mRNA are Higher in Shoot Apices and Flowers than Mature Leaves

We examined the levels of transcripts of each of these genes in shoot apices, mature leaves and flower buds by quantitative real-time RT-PCR (Fig. 2A). Transcripts of AtMAP65-2 and AtMAP65-3 accumulated at relatively higher levels in shoot apices and flower buds containing proliferating cells than in mature leaves, and AtMAP65-1 was highly expressed in all tissues (Fig. 2A).

Figure 2.

Involvement of AtMAP65-1, AtMAP65-2 and AtMAP65-3 in cell division. (A) Expression of genes for AtMAP65-1, AtMAP65-2 and AtMAP65-3 in the indicated organs of Arabidopsis plants. Quantitative real-time RT-PCR was performed to determine the levels of transcripts of genes for AtMA P65s with specific primers for each respective gene. RNA was isolated from the shoot apices of 9-d-old plants and mature leaves and flower buds of 36-d-old plants as previously reported in references 15 and 16. Quantitative RT-PCR data were normalized by reference to the gene for β-tubulin. Error bars indicate SD (n = 3). (B) Comparison of the gross morphology of the atmap65-1-1 atmap65-2-1 (d), atmap65-1-1 atmap65-3-2 (f) and atmap65-2-1 atmap65-3-2 (g) double mutants with that of wild type (a) and each single mutant (b, c and e). All mutant plants had the Col-0 background. Plants were grown for 14 days on plates. Scale bars: 1 cm. (C) Longitudinal root sections from 5-d-old seedlings of wild-type (a), atmap65-3-2 (b), atmap65-1-1 atmap65-3-2 (c) and atmap65-2-1 atmap65-3-2 (g) plants. Roots were stained with propidium iodide (5 µg/mL) and observed by confocal microscopy. Cells in these photographs were located about 50 µm from the median longitudinal axis of the root. Bars: 20 µm.

Mutation of AtMAP65-1 or AtMAP65-2 Enhances the Cytokinetic Defect of the atmap65-3 Mutant

We performed phenotypic analysis of atmap65-1, atmap65-2 and atmap65-3 mutants and double mutants. The atmap65-1-1 allele (SALK_006083) corresponds to the AtMAP65-1 gene with a T-DNA insert in the fourth exon and the atmap65-2-1 allele (GK-849D05) corresponds to the AtMAP65-2 gene with a T-DNA insert in the fifth intron. The map65-3-1 (SALK_022166) and atmap65-3-2 (SALK_118865) alleles correspond to the AtMAP65-3 gene with T-DNA inserts in the second exon and the fifth exon, respectively.

The atmap65-3-1 and atmap65-3-2 mutant plants had short and aberrant expanded roots with cytokinetic defects the same as the previously reported atmap65-3/ple mutant alleles^17,18 (Fig. 2B(e)). As both atmap65-3-1 and atmap65-3-2 were transcriptional null alleles exhibiting the same phenotype, we show only the phenotype of the atmap65-3-2 allele here. Unlike the morphology of atmap65-3 mutants, the morphology of atmap65-1-1 and atmap65-2-2 mutant plants was indistinguishable from that of wild-type plants. In addition, the atmap65-1-1 atmap65-2-1 double mutant had no distinctive phenotype affecting its growth and development (Fig. 2B(a–d)). The dwarfism of atmap65-1-1 atmap65-3-2 and atmap65-2-1 atmap65-3-2 double mutants were, however, more severe than that of the atmap65-3-2 single mutant (Figs. 2B(e–g)). The roots of atmap65-1-1 atmap65-3-2 and atmap65-2-1 atmap65-3-2 double mutants were also shorter than that of atmap65-3-2 (Fig. 2B(e–g)). Furthermore, the roots in these double mutants had more severe cytokinetic defects, with swollen cells having incomplete cell plates (Fig. 2C). When we counted the cells with incomplete cell walls in longitudinal root sections of these mutants, the percentage of cells with incomplete cell walls in the atmap65-1-1 atmap65-3-2 and atmap65-2-1 atmap65-3-2 double mutants [33 ± 6.0% (n = 5) and 33 ± 5.1% (n = 4), respectively] were higher than in the atmap65-3-2 single mutant [23 ± 3.7% (n = 4)]. These results suggest that AtMAP65-1 and AtMAP65-2 function redundantly with AtMAP65-3 downstream of MPK4 in plants. Further investigation on the functions of phosphorylated MAP65s will provide insight into the molecular mechanisms for cell plate formation, especially phragmoplast expansion, during cytokinesis.

Addendum to: Kosetsu K, Matsunaga S, Nakagami H, Colcombet J, Sasabe M, Soyano T, et al. The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana. Plant Cell. 2010;22:3778–3790. doi: 10.1105/tpc.110.077164.