MOR1/GEM1 has an essential role in the plant-specific cytokinetic phragmoplast

Abstract

MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase arrays of cortical microtubules in plant cells. Here we show that MOR1 binds microtubules in vivo, localizing to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. Genetic complementation of the cytokinesis-defective gemini pollen 1-1 (gem1-1) mutation with MOR1 shows that MOR1 (which is synonymous with the protein GEM1) is essential in cytokinesis. Phenotypic analysis of gem1-1 and gem1-2, which contains a T-DNA insertion, confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both the gem1-1 and gem1-2 mutations cause the truncation of the MOR1/GEM1 protein. In addition, the carboxy-terminal domain of the protein, which is absent in both mutants, binds microtubules in vitro. Our data show that MOR1/GEM1 has an essential role in the cytokinetic phragmoplast.

Figure 1

MOR1/GEM1 decorates microtubules and oryzalin induced tubulin aggregates. Arabidopsis suspension culture protoplasts or cells were double stained for tubulin and MOR1 at various stages of the cell cycle. Images show anti-tubulin (green, left panels) and anti-MOR1/CT (red, central panels) fluorescence; yellow colouration in merged images (right panels) represents co-localisation. a-b, Arabidopsis protoplasts. a, interphase cortical array. b, Oryzalin treated protoplasts (2 h at 25°C). c-e, Arabidopsis cells c, preprophase band d, metaphase spindle. e, late anaphase spindle. f, phragmoplast.

Figure 2

gem1-2 shows a cytokinesis defective phenotype. Isolated pollen at early bicellular stage was fixed and stained with DAPI. Bright field (top panels) and epi-fluorescence (bottom panels) images of wild-type and gem1-2. Wild-type pollen shows a typical curved cell wall, but gem1-2 like gem1-1, pollen fail in cytokinesis or produce complex, branching walls (arrowheads) after completion of karyokinesis.

Figure 3

MOR1/GEM1 gene and protein structures. a, Diagram of MOR1/GEM1 exon structure (black bars) showing the positions of the base changes in gem1-1, the position of the T-DNA insertion in gem1-2, and the base changes in mor1-1 and mor1-2. b, Diagram representing the motif content of MOR1/GEM1. Positions of the mor1 (see ref. 1) and gem1 mutations are shown, together with the expected GEM1 truncation products and the length of the expressed C-terminal fragment used for antibody generation and microtubule co-sedimentation assays. Motifs were identified using bioinformatic algorithms located on web-based servers: http://www.ch.embnet.org/software/TMPRED_form.html; http://psort.nibb.ac.jp; http://smart.embl-heidelberg.de/smart/; http://www.embl-heidelberg.de/~andrade/papers/rep/;.

Figure 4

MOR1/GEM1 C-terminus binds to pig-brain microtubules. Co-sedimentation of recombinant MOR1/GEM1 (aa 1123-1978) with microtubules analysed by SDS-PAGE and stained with Coomassie. S, supernatant. P, microtubule pellet.