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2005
LSU-HHMI Summer Undergraduate Research Program |
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Celeste Gary and David Longstreth, Biological Sciences
Increasing Salt Concentration Stimulates Changes in Secreted
Proteins by Suspension Cells from Alternanthera philoxeroides
Plants and cell-suspension cultures of Alternanthera philoxeroides
(alligator weed) tolerate significant increases in salinity.
Our goal was to learn more about the production of a secreted,
22 kD polypeptide from suspension cultures of A. philoxeroides
that is stimulated by exposure to 200 mM NaCl. Heterotrophic
suspension cells, not previously exposed to high salinity, were
sub-cultured in nutrient media (control) or nutrient media with
varying concentrations of NaCl or KCl for various periods of
time. After removal of cells, media proteins were precipitated
with 60% (NH4)2SO4, and then dialyzed in dilute buffer. Cells
were ground in buffer to extract soluble proteins for comparison.
Media and cell proteins were separated by gel electrophoresis
(SDS-PAGE). For seven-day old cultures a polypeptide, approximately
22 kD in size, was found in high-salt (200 mM NaCl) media but
not in control media.
The 22 kD polypeptide was not present in SDS-PAGE of soluble
cell proteins grown in either the control or high-salt treatments.
In fact, SDS-PAGE proteins of the control and high-salt soluble
cell proteins were the same. The presence of the 22 kD polypeptide
was examined at different times after transfer to 200 mM NaCl
media. The intensity of the 22 kD polypeptide was weak on days
two through four. After day four, the intensity of the 22 kD
polypeptide increased through day six but then decreased on
day ten.
The effect of 200 mM KCl on the SDS-PAGE profile for media proteins
was also examined. The 22 kD polypeptide was present in the
media containing KCl indicating it is not just stimulated by
NaCl. Suspension cells were also cultured in 50 mM and 100 mM
NaCl for seven days. Since the 22 kD polypeptide was more intense
in the 200 mM NaCl than at lower NaCl media concentrations,
stimulation of the 22 kD polypeptide appeared to be affected
by NaCl concentration. We examined the possibility that the
22 kD polypeptide was present in the cell wall of control cells
and was only released by high NaCl concentration. Control cultures
grown for seven days were treated with 200 mM NaCl for five
minutes. The 22 kD polypeptide was not present in this media.
Also, filtered cells from control cultures grown for seven days
were exposed to 100 mM CaCl2 and 90 mM sucrose for 30 minutes.
Although this treatment did release proteins from the cells,
the 22 kD polypeptide did not appear to be among them. Preliminary
data indicates that the 22 kD polypeptide may be related to
osmotin, a protein that appears to be part of a plant signal
pathway stimulated by stress. |
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