Electron tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis thaliana

Abstract

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.

Vesicle Trafficking during Somatic Cytokinesis

“…The darkstained vesicles were also found in the vicinity of the Golgi apparatus, and it was concluded that they were Golgi derived. Prevacuolar compartment/multivesicular bodies (MVBs) or late endosomes were detected around the cell plate at its maturation phase, accompanied by an increased appearance of clathrin-coated vesicles (Otegui et al, 2001;Otegui and Staehelin, 2004;Segui-Simarro et al, 2004;Segui-Simarro and Staehelin, 2006). The observation that Arabidopsis (Arabidopsis thaliana) Golgi stacks double before mitosis (Segui-Simarro and Staehelin, 2006) supports the capacity for substantial vesicle secretion during plant cytokinesis.…”

“…Indeed, ultrastructural analysis has revealed a close association of vesicles with the phragmoplast microtubules (Kakimoto and Shibaoka, 1987;Samuels et al, 1995), the integrity of which is essential for cell plate vesicle delivery (Steinborn et al, 2002). Proteins connecting vesicles to phragmoplast microtubules have been visualized during endosperm cellularization and pollen cytokinesis (Otegui et al, 2001;Otegui and Staehelin, 2004). The kinesin AtPAKRP2 is so far the best candidate motor protein to transport the cell plate-building vesicles to the division plane (Lee et al, 2001), although genetic data for this are lacking.…”

“…While the early steps of cytokinesis have been elaborately studied, both genetically (Nacry et al, 2000;Sollner et al, 2002;Jurgens, 2005aJurgens, , 2005bKonopka et al, 2006; and structurally (Samuels et al, 1995;Otegui and Staehelin, 2004;SeguiSimarro et al, 2004;Austin et al, 2005;Segui-Simarro and Staehelin, 2006), few data have been generated regarding the final steps of cytokinesis (Fig. 1B).…”

MIKC* MADS Domain Heterodimers Are Required for Pollen Maturation and Tube Growth in Arabidopsis

“…Substitution and subsequent embedding of samples in Epon resin (Ted Pella) were carried out as described previously by Otegui and Staehelin (2004). Eighty-to 100-nm sections were cut and stained with uranyl acetate (8% in 50% ethanol) and Reynolds lead citrate (Reynolds, 1963) before imaging with a CM120 transmitting electron microscope (Philips).…”

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

“…Electron tomography, which is a powerful electron microscopic technique for getting a nano-scale three-dimensional analysis, has been used in the ultrastructural studies of cytokinesis of land plants (Otegui and Staehelin 2004, Segui-Simarro et al 2004, Haas and Otegui 2007. This technique will be also useful for analysis of tiny PD structure, in order to investigate their ultrastructure and the process of their formation.…”