Two Arabidopsis phragmoplast-associated kinesins play a critical role in cytokinesis during male gametogenesis

Abstract

In plant cells, cytokinesis is brought about by the phragmoplast. The phragmoplast has a dynamic microtubule array of two mirrored sets of microtubules, which are aligned perpendicularly to the division plane with their plus ends located at the division site. It is not well understood how the phragmoplast microtubule array is organized. In Arabidopsis thaliana, two homologous microtubule motor kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, localize exclusively at the juxtaposing plus ends of the antiparallel microtubules in the middle region of the phragmoplast. When either kinesin was knocked out by T-DNA insertions, mutant plants did not show a noticeable defect. However, in the absence of both kinesins, postmeiotic development of the male gametophyte was severely inhibited. In dividing microspores of the double mutant, microtubules often became disorganized following chromatid segregation and failed to form an antiparallel microtubule array between reforming nuclei. Consequently, the first postmeiotic cytokinesis was abolished without the formation of a cell plate, which led to failures in the birth of the generative cell and, subsequently, the sperm. Thus, our results indicate that Kinesin-12A and Kinesin-12B jointly play a critical role in the organization of phragmoplast microtubules during cytokinesis in the microspore that is essential for cell plate formation. Furthermore, we conclude that Kinesin-12 members serve as dynamic linkers of the plus ends of antiparallel microtubules in the phragmoplast.

Figure 1.

Mutations at the Kinesin-12A and Kinesin-12B Loci. (A) Diagrammatical representation of the Kinesin-12A and Kinesin-12B gene structures. Introns are shown as lines, and exons are shown as boxes. Positions of the T-DNA mutational insertions of kinesin-12a-1, kinesin-12a-2, kinesin-12b-1, and kinesin-12b-2 are shown at top of the diagrams. (B) The homozygous double mutant of kinesin-12a-1 and kinesin-12b-2 produced significantly fewer seeds in siliques. Red arrows point to ovule positions where no seeds were found. (C) Quantification of seed production in wild-type and mutant siliques. The y axis represents the percentage of ovule positions with seeds. Plants bearing homozygous single mutations at either locus produced similar numbers of seeds as their wild-type counterparts. The homozygous double mutant and its F1 progeny produced ∼50% fewer seeds. Genotypes of the plants are as follows: 12A 12B for plants with wild-type alleles at both Kinesin-12A and Kinesin-12B loci; 12a 12B for plants with a homozygous mutation at the Kinesin-12A locus; 12A 12b for plants with a homozygous mutation at the Kinesin-12B locus; 12a 12b for the homozygous double mutant; and 12a 12b (F1) for the F1 progeny of 12a 12b.

Figure 2.

The Double Mutant Failed to Produce Male Gametes. (A) In the wild type (a), a mature pollen grain contains two sperm and one vegetative nucleus. The sperm nuclei (arrows) and the vegetative nucleus (arrowhead) were revealed by DAPI staining. In (b), a differential interference contrast image shows the pollen appearance. Transmission electron microscopy images ([c] and [d]) show the vegetative nucleus (VN) and one sperm cell (arrow) in the pollen cytoplasm. Note that the sperm cytoplasm was physically separated from the pollen cytoplasm by a barrier (arrows in [d]). The other sperm cell was not included in this section. Bars = 10 μm in (b) for (a) and (b), 4 μm in (c), and 1 μm in (d). (B) In the double mutant, a defective pollen grain failed to produce sperm. DAPI staining (a) showed two loosely packed DNA masses (arrowheads) resembling the vegetative nucleus. A differential interference contrast image of this pollen is shown in (b). Transmission electron microscopy images ([c] and [d]) show two similar nuclei (N) suspended in the pollen cytoplasm. Note that between the nuclear envelopes of the two nuclei (arrowheads), there was no barrier as seen in ([A], [d]). Bars = 10 μm in (b) for (a) and (b), 4 μm in (c), and 1 μm in (d). (C) Quantification of pollen grains in the categories of two sperm nuclei plus one vegetative nucleus (2+1), two identical nuclei (1+1) or one DNA mass (1), and shrunken appearance (s). The y axis represents the proportion of pollen grains in each category. Pollen grains in the three categories were quantified in the wild type (12A/12A; 12B/12B), single mutants (12a/12a; 12B/12B and 12A/12A; 12b/12b), various heterozygous double mutants (12A/12a; 12B/12b, 12a/12a; 12B/12b, and 12A/12a; 12b/12b), and the homozygous double mutant (12a/12a; 12b/12b).

Figure 3.

Alteration of At Kinesin-12 Expression by T-DNA Insertional Mutations. (A) Absence of the Kinesin-12A and/or Kinesin-12B transcripts in single and double homozygous mutants by RT-PCR. The transcripts were detected in the wild type (12A/12A; 12B/12B), and either transcript was detected in mutants bearing either one or two copies of the wild-type Kinesin-12A or Kinesin-12B gene (12a/12a; 12B/12B, 12A/12A; 12b/12b, 12a/12a; 12B/12b, and 12A/12a; 12b/12b). The At1g13320 transcript encoding protein phosphatase 2A (PP2A) was used as a positive control. (B) Localization of At Kinesin-12A in dividing microspores of the wild type (12A 12B) and the double mutant (12a 12b). The At Kinesin-12A signal is pseudocolored green, and DNA is pseudocolored red. While in the wild type, microspore-specific signals (white arrowheads) were detected between the vegetative nucleus (blue arrows) and the generative nucleus (purple arrows), no such signal was detected in the microspore of the double mutant. The peripheral signal was due to the autofluorescence of the pollen coat. Bars = 5 μm.

Figure 4.

Comparisons of Microtubule Organization and Cell Plate Development during the First Mitotic Cell Division in the Microspore. Microtubules are pseudocolored green, and DNA is pseudocolored blue. (A) In wild-type microspores (a), upon the completion of mitosis, microtubules were organized into an antiparallel array between two DNA masses. A typical phragmoplast microtubule array (b) had a dark line in the middle. The phragmoplast microtubule array (c) appeared in a barrel-like shape at this late stage of cytokinesis. (B) In defective microspores of the homozygous double mutant, microtubules failed to be organized into an antiparallel phragmoplast array. Microtubules (a) polymerized into bundles between two DNA masses after mitosis. More microtubules were formed between two reforming nuclei (b), and they did not appear to have a dark line by tubulin immunofluorescence in the middle (c). Aggregates/bundles of microtubules ([d] and [e]) remained to be associated with one nucleus toward the periphery. Bar = 5 μm.

Figure 5.

The Cell Plate Failed To Be Formed in Defective Microspores. (A) Localization of the cell plate marker KNOLLE in developing pollen grains of the wild type and the double mutant. In a wild type (12A 12B) microspore, the developing cell plate marked by the syntaxin-like protein KNOLLE (arrowheads) was formed in the middle region of the phragmoplast. A dark midline was clearly seen by tubulin immunofluorescence (arrowheads). In defective microspores of a homozygous double mutant (12a 12b), microtubules failed to be organized into a phragmoplast array with a dark midline. KNOLLE accumulated around microtubules in a diffuse fashion. The peripheral signal was due to the autofluorescence of the pollen coat. Bars = 10 μm. (B) Callose accumulation in the wild type and the double mutant. In the wild type, callose (small arrowheads), labeled by aniline blue, appeared between the reforming vegetative nucleus (large arrowhead) and the generative nucleus (arrow) stained by DAPI. The completion of cytokinesis left a callose-rich cell plate (arrowheads) separating the cytoplasms of the generative cell and the vegetative cell (top right). In defective pollen grains in the double mutant, callose accumulated as a large aggregate at the cell cortex (small arrowhead), while two identical nuclei (large arrowheads) were positioned away from the aggregate. Such a callose-rich aggregate (arrowhead) was not organized in a cell plate–like configuration at the cell cortex (bottom right). Bar = 10 μm.

Figure 6.

Models of the Function of Kinesin-12. (A) Kinesin-12A/B and their putative anchoring factor(s) form a protein complex that interacts with the plus ends of phragmoplast microtubules located in the middle region. They function in translocating newly polymerized microtubule segments and allow the plus ends to be stably located in the middle region. (B) The presence of Kinesin-12A/B allows the formation and maintenance of the antiparallel phragmoplast microtubule array. Consequently, successful cytokinesis brings about the cell plate (red), which separates the generative cell from the vegetative cytoplasm. The generative cell undergoes mitosis to produce two sperm cells. The absence of Kinesin-12A/B causes microtubules to be bundled together with mixed polarities. Consequently, materials for cell plate formation do not accumulate in the middle region. Ultimately, two nuclei are suspended in the vegetative cytoplasm. Microtubules are shown in green, and nuclei are shown in blue.