The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment

Abstract

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-alpha-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

Fig. 1.

Pollen development in ProNTM19-GFP::TUA6 transgenic plants. DAPI-stained spores/pollen are viewed with DIC (upper panel) or in epifluorescence (lower panel) from tetrad (A), early microspore just after callose dissolution (B), microspore before nuclear migration (C), after the first equatorial nuclear migration (D), during (E) and after the second polar migration (F, G), during (H–K) and after microspore division (L–O), and mature pollen (P) stages. Images left or right represent views from the radial wall or germ cell pole (GP) (C–F, I). Black arrowheads indicate apertures and filled circles are equivalent to marks on the diagram as an orientation reference. White arrows indicate the germ cell wall attached to the pollen wall. Size bars depict 10 μm.

Fig. 2.

Live spores and pollen from ProNTM19-GFP::TUA6 transgenic line. Fresh spores and pollen viewed with DIC (grey) or with CLSM (green). (A, B) polarizing microspores, (C–J) microspores during mitosis, (K) early and (L) mid-bicellular pollen, (M, N) mature pollen. Microspores after equatorial nuclear migration (A) and after polar nuclear migration (B). Microspores are viewed as a single optical section (centre) or as projections of z-series (right) from the GP. (C–F) Microspores undergoing nuclear division are viewed from the GP (C) or from the radial wall (D–F). Mitotic spindles at metaphase (D), anaphase (E), and telophase (F). Images are single optical sections (C, E, F) or a projection of a z-series (D). (G–J) Microspores undergoing cytokinesis are viewed from the GP (G, I) or from radial wall (H, J). The phragmoplasts at early (G, H) or at later (I, J) stages. Images are single optical sections. (K–O) Fresh spores after microspore mitosis and mature pollen freshly dehisced or incubated in germination media are viewed. Early bicellular (K), mid-bicellular spores (L), mature pollen (M, N) or germinated pollen at three successive minute intervals, top to bottom (O). Images are single optical sections. Black arrowheads indicate apertures seen from the future GP. White arrowheads indicate a MT-rich tail connecting the germ cell and vegetative nucleus. v, Microspore vacuole; n, microspore nucleus; gn, germ cell nucleus; vn, vegetative nucleus. An orientation schematic for the images is shown. The white line with double arrow heads represents length measured for metaphase (D) and anaphase to telophase (E, F) spindles and for the phragmoplast (J).

Fig. 3.

Verification of the link between mutant phenotype and TMBP200 expression in TMBP200 RNAi plants. (A–H) Fresh spores from wild type (A) or RNAi lines (B–H) are viewed with DIC. Black arrowheads indicate the position of walls. Three arrowheads (F–H) indicate the complex profile of walls and asterisks (H) indicate three nuclei. (I–P) Callose staining of walls with aniline blue. White arrowheads indicate aniline blue stained walls. Fixed spores at early bicellular (I–L) and mature pollen (M–P) stages viewed by epifluorescence. In contrast to wild-type pollen (I, M) showing a lens-shaped callose wall only at the early bicellular stage, mutant pollen from RNAi lines display complex and persistent callose walls (J–L, N–P). (Q–S) Transmission electron micrographs of pollen at the bicellular stage. While wild-type pollen shows a smooth and hemispherical barrier between the two daughter nuclei (Q), mutant pollen displays highly complex walls (R, S) similar to those observed in gem1. Black arrowheads outline the walls. (T) RT-PCR analysis using mature pollen RNA and TMBP200-specific primers. A tobacco actin gene, NtACTIN, was used as the internal control to show an equivalent amount of cDNA templates were used between samples. C5 control plants (lanes 1, 2) and two intermediate TMBP200 RNAi plants, line 18 (lanes 3, 4) and line 20 (lanes 5, 6), were analysed in duplicate from separately prepared RNA samples.

Fig. 4.

Pollen development in TMBP200 RNAi lines. Fixed spores were stained with DAPI and viewed by DIC (upper panels) or epifluorescence (lower panels). Microspores undergoing mitosis with impaired polarity (A–D), microspore showing lagging chromosomes (E–H) or misaligned chromosomes at meta-anaphase (H–L), pollen after mitosis showing two equal nuclei at the pole (M) or in the centre (N) or micronuclei (O, P) at the bicellular stage, binucleate (Q), fusion nucleate (R) or aborted pollen (S) at the mature pollen stage. Images are shown in a radial view (A–P) except for those at the mature pollen stage (Q–S). An orientation schematic for the images is shown.

Fig. 5.

Mitotic and cytokinetic apparatus in TMBP200 RNAi lines. Spindles (A–F) and phragmoplasts (G–L) from microspores undergoing aberrant mitosis or cytokinesis were viewed with DIC (upper panel) and with CLSM (lower panel). Images are shown in radial view (A, D, E, F, G, J, K), GP view (B, C, H, I) or at an angle in between (L). Arrowheads (B, C, H, I, L) indicate positions of pollen apertures. Mutant spindles represent bipolar spindles 90° rotated (A, B, C) and multipolar spindles (D, E, F). Mutant phragmoplasts represent single but misoriented (G, H) and complex branched or fragmented forms (I, J, K, L). An orientation schematic and wild-type spindle and phragmoplast images are shown for radial (R) and polar (P) views.