SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4kβ1/pi4kβ2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.

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The three-dimensional architecture of syncytial-type cell plates in the endosperm of Arabidopsis has been analyzed at approximately 6-nm resolution by means of dual-axis high-voltage electron tomography of high-pressure frozen/freeze-substituted samples. Mini-phragmoplasts consisting of microtubule clusters assemble between sister and nonsister nuclei. Most Golgi-derived vesicles appear connected to these microtubules by two molecules that resemble kinesin-like motor proteins. These vesicles fuse with each other to form hourglass-shaped intermediates, which become wide (approximately 45 nm in diameter) tubules, the building blocks of wide tubular networks. New mini-phragmoplasts also are generated de novo around the margins of expanding wide tubular networks, giving rise to new foci of cell plate growth, which later become integrated into the main cell plate. Spiral-shaped rings of the dynamin-like protein ADL1A constrict but do not fission the wide tubules at irregular intervals. These rings appear to maintain the tubular geometry of the network. The wide tubular network matures into a convoluted fenestrated sheet in a process that involves increases of 45 and 130% in relative membrane surface area and volume, respectively. The proportionally larger increase in volume appears to reflect callose synthesis. Upon fusion with the parental plasma membrane, the convoluted fenestrated sheet is transformed into a planar fenestrated sheet. This transformation involves clathrin-coated vesicles that reduce the relative membrane surface area and volume by approximately 70%. A ribosome-excluding matrix encompasses the cell plate membranes from the fusion of the first vesicles until the onset of the planar fenestrated sheet formation. We postulate that this matrix contains the molecules that mediate cell plate assembly.