[28]
Ultracellular
Localization of ARG1 in Arabidopsis COLUMELLA CELLS
A.K. Spence, N.S. Kumar, R.E. Edelmann, and
J.Z.
Kiss. Dept. of Botany, Miami Univ., Oxford, OH.
Gravitropism
is the process by which plants
direct organ growth in response to gravitational stimuli. In the roots,
gravity
is initially signaled by the movement of starch-containing amyloplasts
located
within the gravity perceiving cells. These cells, known as statocytes,
collectively make up the columella located within the root tip. The
peripheral membrane protein ARG1 has been found to play a role in the
early
phase of gravitropism (signal-transduction) through its presence in the
columella of the root. Previous studies at the confocal level have
found ARG1
to be associated with cellular compartments of the elongation zone and
root cap
columella cells including the Golgi stacks, vesicles, endoplasmic
reticulum,
and the plasma membrane. This association with compartments of the
secretory
pathway supports the idea that ARG1 is involved in gravitational
signal-transduction, possibly aided by its interaction with the
cytoskeleton of
the statocytes. Our current research aims to localize ARG1
at the ultracellular level using higher resolution transmission
electron
microscopy. Both light and dark grown Arabidopsis seedlings of an ARG-GFP line
were prepared for TEM using two different chemical fixation protocols. The first procedure fixed the specimens in
paraformaldehyde and glutaraldehyde while the second procedure utilized
a
mixture of glutaradehyde and osmium tetroxide as to fix and provide en
bloc
staining simultaneously. The seedlings were then infiltrated and
flat-embedded
with LR White resin. Ultra-thin sections
of the embedded specimens were immuno-labeled with primary anti-GFP
antibody
and secondary gold anti-Rabbit IgG antibody and then imaged with a JOEL
transmission electron microscope. Using the data obtained from the
immuno-labeling, we hope to determine the specific membranes and
organelles
with which ARG1 interacts and to ascertain the nature of the protein’s
association with the cytoskeleton. Localizing ARG1 at the ultracellular
level
will provide further insight into the signal-transduction phase of
gravitropism.
(Supported
by the Miami U. Undergraduate Summer Scholars Program and
NASA.)
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