272 J.M. Seguí-Simarro et al.
the network and the generation of new dumbbell-shaped vesicles that serve
as new nucleators for cell plate assembly. The resulting structure is a plate-
like, labyrinthic membranous network consisting of vesicular and cisternal
domains interconnected by thin, short tubules (Figs. 4B, 7B).
One of the valuable aspects of electron tomography is the ability to quan-
tify the structures that have been reconstructed. In the Seguí-Simarro et al.
(2004) study, the tomographic data were used to calculate both the unit cell
plate surface area and the unit cell plate volume changes during the differ-
ent stages of cell plate assembly. Since publication of that study, we have been
informed that there was an error in the algorithm used to calculate the vol-
ume of the objects with irregular morphologies (D. Mastronarde, personal
communication). We have now recalculated all of the volumetric data of our
tomographic database using the corrected algorithm (Fig. 8A). Compared to
the original data (Fig. 11 of Seguí-Simarro et al. 2004), all of the recalcu-
lated cell plate volumes perμm^3 of tomogram volume are threefold higher. In
terms of understanding cell plate assembly, the only significant change relates
to the events associated with the assembly of the tubulo-vesicular network.
In particular, the final tubulo-vesicular cell plate has a volume similar to the
volume of all of the vesicles that contributed to its assembly as determined
by the cell plate surface area calculations. This lack of cell plate volume re-
duction compared to the volume reduction of the dumbbells may be due to
having only a fraction of the vesicles that contribute to cell plate growth con-
verted into shrunken dumbbell-type intermediates prior to their fusion with
the cell plate. Furthermore, because the synthesis of callose by cell plate lo-
cated enzymes commences before the breakdown of the CPAM (Samuels et al.
1995), any cell plate volume reduction caused by the dynamin motor pro-
teins may be masked by the accumulation of this polysaccharide in the cell
plate lumen.
At the end of the tubulo-vesicular cell plate growth phase (Fig. 8B), the
cellplatecontainsmorethanenoughmembranetoproducethenewcellwall
(Seguí-Simarro et al. 2004). This state of the cell plate appears to produce
a maturation signal that results in the cessation of new vesicle delivery to the
cell plate region by causing a breakdown of the CPAM (Figs. 2D, 4C) and the
disassembly the solid phragmoplast MTs that guide the vesicles to that region
(Figs. 2D, 3D; Samuels et al. 1995; Ueda et al. 2003; Seguí-Simarro et al. 2004;
Austin et al. 2005; Szechynska-Hebda et al. 2006). As this breakdown occurs
(transitional stage of cell plate assembly) the CPAM components are moved to
the periphery of the tubulo-vesicular cell plate (Figs. 2D, 4C), and a new set of
MTs become stabilized in a ring around the cell plate margins (Figs. 2E, 3E).
In this manner, the new vesicles are directed to the now centrifugally grow-
ing cell plate regions. Mechanistically, centrifugal cell plate growth resembles
growth of the initial cell plate in the solid phragmoplast, and this growth
ceases when the cell plate tubules start fusing with the plasma membrane
(Fig. 4D; Samuels et al. 1995; Seguí-Simarro et al. 2004).