Plant Physiol. (1998) 116: 223-229
Ethylene-Mediated Phospholipid Catabolic Pathway in
Glucose-Starved Carrot Suspension Cells1
Soo Hyun Lee,
Hyun Sook Chae,
Taek Kyun Lee,
Se Hee Kim,
Sung Ho
Shin,
Bong Huey Cho,
Sung Ho Cho,
Bin G. Kang, and
Woo Sung Lee*
Department of Biology, Sung Kyun Kwan University, Suwon 440-746,
Korea (S.H.L., T.K.L., S.H.K., W.S.L.); Department of Biology, Yonsei
University, Seoul 120-749, Korea (H.S.C., B.G.K.); Agency
for Defense Development, Yuseong 305-600, Korea (S.H.S.); Department
of Biology, Suwon University, Suwon 445-743,
Korea (B.H.C.); and Department of Biology, Inha University, Inchon
402-751, Korea (S.H.C.)
 |
ABSTRACT |
Glucose (Glc) starvation of
suspension-cultured carrot (Daucus carota
L.) cells resulted in sequential activation of phospholipid catabolic
enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at
the early part of starvation, and these activities were followed by
-oxidation and the glyoxylate cycle enzymes in order. The activity
of PLD and lipolytic acyl hydrolase was further confirmed by in
vivo-labeling experiments. It was demonstrated that Glc added to a
medium containing starving cells inhibited the phospholipid catabolic
activities, indicating that phospholipid catabolism is negatively
regulated by Glc. There was a burst of ethylene production 6 h
after starvation. Ethylene added exogeneously to a Glc-sufficient
medium activated PLD, indicating that ethylene acts as an element in
the signal transduction pathway leading from Glc depletion to PLD
activation. Activation of lipid peroxidation, suggestive of cell death,
occurred immediately after the decrease of the phospholipid
degradation, suggesting that the observed phospholipid catabolic
pathway is part of the metabolic strategies by which cells effectively
survive under Glc starvation.
| |
INTRODUCTION |
Once C sources become limited, plant cells actively adjust their
metabolic strategy to cope with adverse growth conditions. Starved
cells generally exhibit a decrease in respiratory capacity and scavenge
alternative C sources from cellular constituents such as carbohydrates,
lipids, proteins, or other cellular materials (Journet et al., 1986
;
Roby et al., 1987; Brouquisse et al., 1991). It is well established
that plant leaves or suspension cells sacrifice their cellular membrane
phospholipids to generate fatty acids and their downstream metabolites
for ATP production once they face Glc or Suc starvation (Thompson,
1988; Graham et al., 1994). The activity of fatty acid -oxidation
increased in Glc-starved maize root tips (Dieuaide et al., 1992),
indicating that fatty acid degradation occurs in response to
starvation. Several studies have demonstrated that the glyoxylate cycle
enzymes are also induced in leaves or suspension cells undergoing Glc
starvation (Kudielka and Theimer, 1983; Gut and Matile, 1988; Graham et
al., 1992; Lee and Lee, 1996).
In starved cells acetyl-CoA, which is produced by -oxidation, is
mainly funneled into the glyoxylate cycle rather than into the Krebs
cycle, in which C atoms are lost as CO2. These
phospholipid catabolic activities were also observed in senescing
leaves from a number of plant species (Thompson, 1988; Paliyath and
Droillard, 1992). Starvation is also likely to occur in senescing
leaves, from which residual C sources are mobilized into the stem
before leaf death and abscission. It is therefore likely that Glc
starvation resembles leaf senescence, at least in regard to a metabolic
response toward starvation.
In addition to the role as one of the preferred C sources in plants,
Glc is also known to be an important regulator involved in a number of
metabolic processes. Glc represses the transcription of several
photosynthetic genes (Sheen, 1990; Krapp et al., 1993). 
-Amylase
activity was repressed in the presence of Glc in rice cell suspensions
(Yu et al., 1991). Glc appeared to be initially sensed by several
cellular components, including hexokinase (Jang and Sheen, 1997),
allowing the target enzymes to respond to the cellular Glc level. One
of the candidates involved in the signal transduction in response to
Glc starvation is ethylene, a gaseous hormone that plays diverse roles
in many growth and developmental processes, including leaf senescence
(Nooden, 1988). During leaf senescence exogenously applied ethylene has
been shown to hasten several metabolic processes, such as activation of
the many hydrolytic enzymes (Suttle and Kende, 1980; Grbic and Bleeker,
1995). It is therefore expected that ethylene is also involved in the
signal transduction and adaptive response under Glc starvation in
carrot (Daucus carota L.) suspension cultures. This study
demonstrates that phospholipid degradation, a process that is one
aspect of the adaptive response, was mediated by ethylene.
It is known that cells undergoing C starvation initially utilize
cellular starch or Suc, both of which are more readily disposable (Journet et al., 1986). As Glc starvation persists, cells start to
degrade their own membrane phospholipids. It is very likely that
starved cells adopt a precisely controlled phospholipid catabolic pathway by which they manage to sustain their basal metabolic capacity.
Multiple enzymes, such as the various phospholipases and LAH, may
participate in phospholipid degradation in a coordinated manner.
Recently, PLD has received much attention, since it has been found to
be involved in a number of signaling systems in animals and yeast
(Billah, 1993; Exton, 1994).
Even though the participation of PLD in cell signaling has not been
conclusively demonstrated, PLD has also been suggested to be associated
with signal perception in plants (Causier and Millner, 1996; Pappan et
al., 1997; Wang, 1997). G-protein, a membrane-bound signaling element,
is suggested to stimulate PLD in senescing carnation petals (Munnik et
al., 1995). In this study we suggest that PLD is a signaling element
perceiving Glc starvation, and that hydrolysis of the phospholipid head
group, catalyzed by PLD, is the earliest biochemical event involved in
the destruction of the membrane phospholipids under Glc starvation. It
is also suggested that this PLD-initiated phospholipid catabolism may represent a well-controlled adaptive response to Glc starvation.
| |
MATERIALS AND METHODS |
Cell Culture
Carrot (Daucus carota L.) suspension cells, originated
from tap roots, were maintained by weekly subculturing. Detailed
culture conditions and medium compositions were as described by Lee and Lee (1996). To initiate Glc starvation, cells actively growing with Glc
were transferred to the same medium without Glc. At the designated
interval, cells were aseptically harvested and immediately frozen at
80°C. Protein concentration was measured using a Bio-Rad protein
assay kit with BSA as the standard.
Assays of Phospholipase A, PLC, PLD, LAH, -Oxidation, ICL, and
Peroxidation
For the enzyme assays, cells (1 g) were ground in 2 mL of
homogenization buffer (170 mm Tricine-NaOH, pH 7.5, 10 mm KCl, 1 mm EDTA, and 10 mm DTT)
with a prechilled mortar and pestle. The homogenates were centrifuged
at 12,000g for 20 min, and the supernatants were used for
the enzyme assays. The above procedures were performed at 0 to 4°C.
The activity of the enzymes involved in phospholipid degradation was
determined by the detection of the expected reaction products by
ion-exchange chromatography (Dowex-50 WH+) using
labeled (16:0/16:0)-phosphatidylcholine (1.2-dipalmitoyl choline [choline-methyl-14C, 170 mCi/mmol,
Dupont]) as the substrate.
The activity of PLD and LAH was assayed by the method described by
Paliyath et al. (1987). The assay mixture contained 100 mm
K2PO4 buffer, pH 7.5, and
200 µL of crude extracts in a total 0.5 mL of reaction volume. The
reaction was initiated by the addition of 20 µL of substrate, which
was prepared by sonication with 17.7 µm of cold PC and
2.5 µCi of the labeled PC in 1 mL of water. The reaction was carried
out at 30°C for 1 h. The labeled products of PLD and LAH,
choline and glycerophosphocholine, respectively, were extracted from a
reaction mixture by chloroform:methanol (2:1, v/v). Separation of the
products was performed with ion-exchange column chromatography
(Dowex-50 WH+ column). The column was initially
washed with 5 mL of water to elute glycerophosphocholine. Choline
phosphate was eluted by an additional washing with 20 mL of water.
Finally, choline was eluted by 20 mL of 1 m HCl. The
radioactivity of each fraction was determined using a
liquid-scintillation counter (LS 6500, Beckman). Elution profiles were
confirmed in each experiment by the inclusion of a separate column upon
which the standard compounds were loaded.
The activity of -oxidation was assayed for palmitoyl-CoA-dependent
NAD reduction according to the method of Cooper and Beevers (1969). The
reaction mixture (1 mL) contained 130 mm
K2PO4 buffer, pH 7.5, 0.5 mm MnCl2, 3.1 mm DTT,
0.13 mm CoA, 0.14 mm NAD, and 100 µL of
enzyme extracts. The reaction was initiated by the addition of 6.3 mm palmitoyl-CoA, and the reaction rates were measured at
340 nm. Lipid peroxidation was measured by determining the level of
malondialdehyde by the method described by Heath and Packer (1968). ICL
activity was determined according to the method of Franzisket and
Gerhardt (1980).
Assays of PLD and LAH Activities by in Vivo Labeling
PLD activity in cells was determined essentially by the
transphosphatidylation method described by Munnik et al. (1995).
Actively growing cells were prelabeled with 100 µCi
32Pi (Amersham) per milliliter in growth medium
for 5 h to produce 32P-labeled PC within
cells. Cells were washed with an excessive amount of fresh medium to
remove residual 32Pi, and then underwent
starvation for the designated intervals. Harvested cells were incubated
with 0.25% n-butanol in the medium for 10 min, during which
time the transphosphatidylation reaction occurred. Lipids were
extracted, separated by ethyl acetate TLC (ethyl
acetate/iso-octane/HAc/H2O [13:2:3:10, v/v]),
and autoradiographed. The intensity of the PtdBut spot represents PLD
activity.
To measure LAH activity in vivo, cells were prelabeled with 1 µCi per
[14C]choline (Amersham) per 20 mL for 12 h
in growth medium to produce choline-labeled PC in cells. After removing
residual [14C]choline by repeated washings with
an excessive volume of growth medium, cells underwent starvation for
the designated intervals. Cellular choline-labeled
glycerophosphocholine, the reaction product of LAH, was measured by
ion-exchange chromatography as described above.
Determination of Ethylene Level
After carrot cells were transferred into a 100-mL flask containing
30 mL of Glc-free culture medium, the flask was sealed by a silicon cap
for 1 h at the designated intervals, and the ethylene produced was
determined in 1-mL samples by GC (model GC-3BF, Shimadzu, Columbia,
MD).
| |
RESULTS |
Enzyme Activities Associated with Phospholipid Degradation during
Glc Starvation
Carrot suspension cells actively growing with sufficient Glc were
transferred into a Glc-free medium at time 0 to establish Glc
starvation. Cells actively growing with Glc and cells undergoing Glc
starvation, harvested at d 3 or d 6, were used to determine whether
phospholipid degradative enzymes were activated. Extracts from the
whole cells were used to determine enzyme activities. Each enzyme
activity was assigned by the detection of the degradation products of
labeled PC (14C-choline) using ion-exchange
chromatography (see details in ``Materials and Methods''). PLD and
LAH catalyze the hydrolysis of the phospholipid head group and the
fatty acids at both sn-1/2 positions, respectively. These enzymes were active in cells harvested at d 3 (Fig.
1A).
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| Figure 1.
Enzyme activities participating in phospholipid
degradation during Glc starvation. A, Activities of various
phospholipid degradative enzymes in cells harvested at d 1 (1 d before
Glc starvation,  ), d 3 ( ), and d 6 ( ) after starvation. The
levels of glycerophosphocholine, choline phosphate, and choline
represent the activity of LAH, PLC, and PLD, respectively. Experiments
were repeated twice and results were very similar. Representative data
are shown. B, Lipid peroxidation in cells harvested daily after
starvation. Data are means ± se of three
replicates.
|
|
The levels of choline and glycerophosphocholine, the degradation
products of PLD and LAH, respectively, sharply increased by
approximately 5-fold in these cells compared with levels in cells
harvested 1 d before starvation, at which time only the basal
levels were detected. The data in Figure 1A also showed a trend toward
a decrease in PLD and LAH activities in d 6 compared with d 3, indicating that these enzymes were activated at the early part of
starvation, followed by a gradual decrease as starvation continued.
There was no apparent indication of the products of choline phosphate
(Fig. 1A), suggesting that the activity of PLC was negligible, if
present at all, in carrot cells under Glc starvation.
The activity of lipid peroxidation, measured by the detection of fatty
acid-derived malondialdehyde, was at its basal level until d 6, but
dramatically increased at d 7 and thereafter (Fig. 1B), indicating that
peroxidation was not involved in the initial responses to starvation.
The active peroxidation starting from d 7 suggests that cell membranes
are catastrophically degraded to die at this late stage of starvation.
In summary, these results show that PLD, LAH, and lipid peroxidation,
but not PLC, participated actively in the degradation of phospholipids
in carrot cells under Glc starvation. Detailed temporal changes of
these activities were investigated in the following experiments.
Sequential Activation of the Phospholipid Catabolic Enzymes
This PLD/LAH-associated degradation of cellular membranes can be
considered to be a self-digesting event by which starving cells acquire
alternative C sources and energy to sustain metabolic integrity. Our
immediate interest was to see if there was any temporal regulation in
these enzymes; therefore, the activation pattern was monitored using
the cells harvested daily after starvation (Fig.
2). PLD was activated at d 1 and peaked
at d 2, and LAH was activated at d 1 and peaked at d 3 (Fig. 2). These
observations indicate that PLD and LAH participated in phospholipid
degradation under Glc starvation.
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| Figure 2.
Time-course experiments for PLD and LAH. PLD ()
and LAH () activities were measured using cells harvested daily
after starvation. The detailed methods for the enzyme assays are
described in ``Materials and Methods''. Data are means ± se of three replicates.
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To further confirm the participation and temporal activation of PLD and
LAH, these activities were assayed using cells radiolabeled with
32Pi and 14C-choline,
respectively. The PLD assay was performed in vivo by measuring the
formation of PtdBut in the presence of butanol (Munnik et al., 1995).
This method is known to be specific to PLD, which by a
transphosphatidylation reaction forms PtdBut (Liscovitch, 1989; Moehran
et al., 1994). Cells actively growing with Glc were prelabeled with
32Pi to produce
32P-phospholipids. The cells (1 mg) were
harvested daily after starvation and were incubated with
n-butanol. Labeled PtdBut was detected by TLC (Fig.
3A). Data indicated that PLD was
activated at d 1 and peaked at d 2 (Fig. 3A), and these results agree
with the data obtained by the in vitro experiments shown in Figure 2.
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| Figure 3.
Assays of PLD (A) and LAH (B) activity by in
vivo-labeling experiments. A, Cells prelabeled with 32P and
harvested daily after starvation were used for transphosphatidylation reaction in the presence of n-butanol. Cells (1 mg) were
harvested immediately before starvation, at d 1, d 2, and d 3 (lanes
1-4), and extracted cellular lipids were separated by ethyl acetate TLC. PtdBut and PA are designated by arrows. This experiment was repeated twice and the patterns were very similar. Representative data
are shown. B, Cells were prelabeled with 14C-choline and
divided and treated by either starvation ( ) or nonstarvation for
control (). The level of choline-labeled glycerophosphocholine from
cells harvested after each treatment was measured with an ion-exchange
column. Data are means ± se of three replicates.
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The participation of PLD at the early stages of starvation was further
confirmed by observing the increase in PA, another indicator of PLD
activity, as starvation was prolonged in cells prelabeled with
32Pi before starvation (Fig. 3A). When
14C-choline-labeled cells were assayed for PLD by
measuring the production of labeled choline in starved cells, the assay
was not successful because choline disappeared rapidly upon starvation (data not shown). Instead, these cells were assayed for LAH by monitoring the production of choline-labeled glycerophosphocholine by
ion-exchange column chromatography (Fig. 3B). The results indicated that LAH was activated at d 2 and its activity peaked at d 3, whereas
activity remained low in nonstarved controls. These results are similar
to the results shown in Figure 2.
To determine if the fatty acids thus formed are used for catabolic
purposes, the same cell extracts used for the determination of the
activity of the phospholipid degradation were monitored for the
activity of the -oxidation and the glyoxylate cycle. The activity of
palmitoyl-CoA-dependent NAD reduction and ICL, catalyzing the
conversion of isocitrate into glyoxylate, was measured to represent the
activity of the -oxidation and the glyoxylate cycle, respectively
(Fig. 4). Both activities increased
significantly at d 3. The activity of -oxidation peaked broadly
between d 3 and 6, whereas ICL peaked at d 6, followed by gradual
decreases of these activities. These time-course experiments indicated
that -oxidation preceded the glyoxylate cycle. These results
collectively suggest that cells under Glc starvation formed a
phospholipid catabolic pathway: PLD/LAH 
-oxidation glyoxylate cycle. Even though several intermediate enzymes associated
with the proposed pathway, such as acyl-CoA synthetase, were not
studied, the sequence described here very likely represents the
overall phospholipid catabolic pathway operating during Glc starvation.
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| Figure 4.
Time-course experiments of the activity of
-oxidation () and isocitrate lyase (). Palmitoyl-CoA-dependent
NADH production was used for -oxidation activity. Extracts from
cells harvested daily after starvation were used for the assays. Bar
represents ± se of three replicates. U, Units.
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Ethylene-Mediated Signal Transduction between Glc Starvation and
Phospholipid Degradation
Our next goal was to understand how Glc starvation signals the
induction of the phospholipid catabolic pathway. Glc is thought to be
involved in the control of a number of metabolic processes in such a
way as to maintain a constant level of intracellular C for both
catabolic and anabolic purposes (Jang and Sheen, 1997). It is therefore
reasonable to believe that depletion of Glc somehow signals to activate
the phospholipid catabolic pathway to produce alternative C sources. It
is likely that there are multiple signaling elements with which
cells can sense Glc starvation and in turn activate the phospholipid
degradation. Among the many potential elements involved in these
processes, the phytohormone ethylene was chosen for the study, since it
is known to participate in the response to leaf senescence, during
which extensive hydrolysis of many cellular macromolecules, including
phospholipids, occurs (Nooden, 1988). We measured the amounts of
ethylene produced during the course of starvation to determine at which
point the ethylene level increased (Fig.
5A). Accumulated ethylene in an
airtight culture flask was measured by GC, and a culture containing
nonstarving cells was used as the control. There was a burst of
ethylene production 6 h after the initiation of starvation, and
the amount produced was about three times greater than that of the
control (Fig. 5A).
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| Figure 5.
Involvement of ethylene in sensing Glc starvation.
A, Ethylene production during the course of Glc starvation. Actively
growing cells were transferred into a 100-mL flask containing 30 mL of Glc-free medium to establish Glc starvation at time 0. The measurement of the ethylene level was described in ``Materials and Methods''. The
values shown (open bars) represent the percentage increases against the
levels from nonstarved cells (shaded bars). Nonstarved cells were
initially grown with 3% (w/v) Glc, and a residual concentration of Glc
in the medium was checked throughout the experiments to confirm
nonstarvation (data not shown). Bar represents ± se
of three replicates. B, Ethylene involvement in Glc sensing and Glc
repression of PLD. Ethrel (300 ppm), which releases ethylene, was added
at time 0 to a medium containing actively growing cells with 3% Glc to
initiate a fresh culture (). The same culture without addition of
ethrel was also carried out as a control ( ). To observe Glc
repression of PLD, 3% Glc was added to culture containing cells
starved for 1 d in Glc-deficient medium, and PLD activity was
measured using the Glc-treated cells (). Bar represents mean ± se of three replicates.
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To see if ethylene was involved in conveying the probable signal,
ethrel, a compound that releases ethylene (Warrer and Leopold, 1969),
was added at time 0 to initiate a fresh culture growing in medium
containing sufficient Glc, and activity of PLD was monitored (Fig. 5B).
The activity of PLD increased significantly at d 2 and, furthermore,
the pattern of its activation was largely similar to that in starved
cells shown in Figure 2A. When ethrel was added to Glc-deficient medium
on d 1, there was no further increase of PLD activity at d 2 and 3 (data not shown). As expected, the activity of PLD did not increase on
d 2 when Glc was added to the Glc-deficient medium on d 1 (Fig. 5B).
These results strongly suggest that ethylene is a component in the
signal transduction pathway leading from Glc starvation to activation
of PLD.
| |
DISCUSSION |
This study demonstrated that phospholipid catabolic enzymes were
sequentially activated during Glc starvation in carrot suspension cultures. The fatty acids produced were actively funneled into the
central metabolism, as was evidenced by the activation of -oxidation
and enzymes of the glyoxylate cycle, processes involved in the
conversion of fatty acids into malate and succinate via acetyl-CoA.
The overall phospholipid catabolic pathway determined in this study
represents an active metabolic flow of the available C sources
originated from cellular membranes. Cells undergoing Glc starvation may
have adopted this metabolic strategy as a last resort after consuming
more readily disposable cellular C sources such as Suc or protein. The
decrease in these activities was immediately followed by the activation
of membrane peroxidation (Fig. 1B), a process involved in a
death-causing degradation of membrane lipids (Bowler et al., 1992).
These results strongly imply that the phospholipid catabolism described
here is under strict metabolic control to effectively adapt to the
adverse growth conditions. Since we used only the commercially
available 1.2-dipalmitoyl PC (14C-choline) as the
reaction substrate throughout the experiments, there can be some
deviations from the results presented in this study once we use other
kinds of phospholipids. However, it is unlikely that the kinds of fatty
acid and phospholipid head groups shown here drastically altered the
pattern of the activation of the enzymes involved in the phospholipid
degradation under Glc starvation.
Senescence and several environmental stresses are also known to induce
the degradation of membrane phospholipids. In senescing mung bean
cotyledons, PC was initially degraded into PA, suggesting that PLD was
used for phospholipid degradation (Herman and Chrispeels, 1978). It was
also suggested that PLD and LAH were actively involved during cabbage
leaf senescence (Cheour et al., 1992). Similarly, in 
-irradiated
cauliflower microsomal membranes, PLD and LAH, but not PLC, were
associated with the membrane degradation, and PLD activity was
primarily induced upon -irradiation (Voisine et al., 1993). These
findings together imply that PLD and LAH are widely involved in the
phospholipid degradation under the stresses affecting cellular
membranes. In our Glc-starved carrot suspension cells, both PLD and LAH
were selectively activated, much like the cases described above. Even
though the temporal appearance of PLD and LAH needs to be further
clarified, this study suggested that PLD was activated earlier than LAH
based on their respective peaks (Figs. 2A and 3). These results
strongly suggest that PLD plays critical roles in receiving the signal derived from Glc starvation and in initiating the membrane degradation.
PLD has been considered a participant in a variety of signal
transduction systems in plants (Causier and Millner, 1996; Pappan et
al., 1997) and animals (Liscovitch, 1992). The PLD-mediated hydrolysis
of membrane phospholipids is induced in response to many agents,
including hormones and growth factors, in the mammalian system
(Rothman, 1994). The degradation product of PLD, PA, is known to
participate in many cellular responses, such as networking the other
cellular phospholipases (Liscovitch, 1992). It is thus reasonable to
speculate that Glc starvation activated PLD through an unidentified
signal transduction system in these carrot cells. Considering that PLD
was activated by exogeneously added ethylene in a Glc-sufficient
medium, it is assumed that a signal derived from ethylene was, directly
or indirectly, responsible for PLD activation. Ethylene is known to
activate certain protein kinases to activate target enzymes (Schaller
and Bleecker, 1995; Wilkinson et al., 1995).
How PLD hydrolysis activates the next downstream enzyme is not
understood. PA may control the concentration of the intracellular Ca,
which in turn activates the PLD-downstream enzymes. Alternatively, PLD
hydrolysis of the structural phospholipids may cause membranes to be
destabilized and increase Ca flux across the membrane to activate the
downstream enzymes (Paliyath et al., 1987). It is still possible that
initial membrane disintegration caused by the PLD hydrolysis made the
membrane more physically vulnerable to attacks by LAH or, possibly, by
the other phospholipid degradation enzymes.
This study suggests that the ethylene burst occurring at the very early
stages of starvation is used to perceive the starvation signal and
relays it to PLD. It appears that ethylene initiates the activation of
the hydrolytic enzymes to initiate the adaptive response toward Glc
starvation. Cells undergoing Glc starvation may operate the metabolic
strategy demonstrated here to delay cell death until a better
environment, Glc resupply in this case, becomes available. Since Glc
starvation may occur frequently around plant cells depending on their
source/sink relations during their growth cycle, the metabolic strategy
described here may be widely adopted by plants to cope with Glc or
other C source starvation.
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FOOTNOTES |
1
This work was supported by the Academic Research
Fund (GE 96-212) of the Ministry of Education, Republic of Korea,
awarded to W.S.L. This work was also partly supported by a grant from Korea Science and Engineering Foundation-Hormone Research Center (97-K-3-0401-03) awarded to B.G.K.
*
Corresponding author; e-mail wslee{at}yurim.skku.ac.kr; fax
82-331-290-7015.
Received June 18, 1997;
accepted September 18, 1997.
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ABBREVIATIONS |
Abbreviations:
ICL, isocitrate lyase.
LAH, lipolytic acyl
hydrolase.
PA, phosphatidic acid.
PC, phosphatidylcholine.
PLC, phospholipase C.
PLD, phospholipase D.
PtdBut, phosphatidyl
butanol.
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ACKNOWLEDGMENTS |
We thank Drs. Larry Nooden, Young Joon Oh, and Vicky
Buchanan-Wollaston for critical reading of the manuscript and
suggestions on the study. We also thank Dr. Young Sook Lee for her help
in PLD assays.
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Abstract
Introduction
