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. 1998 Jan;116(1):223–229.

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells1

Soo Hyun Lee 1, Hyun Sook Chae 2, Taek Kyun Lee 1, Se Hee Kim 1, Sung Ho Shin 3, Bong Huey Cho 4, Sung Ho Cho 5, Bin G Kang 2, Woo Sung Lee 1,*
PMCID: PMC35161

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 β-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.

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 β-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 β-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). α-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, β-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 β-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 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).

Figure 1.

Figure 1

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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.

Figure 2.

Figure 2

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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 Methods. Data are means ± se of three replicates.

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.

Figure 3.

Figure 3

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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.

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 β-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 β-oxidation and the glyoxylate cycle, respectively (Fig. 4). Both activities increased significantly at d 3. The activity of β-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 β-oxidation preceded the glyoxylate cycle. These results collectively suggest that cells under Glc starvation formed a phospholipid catabolic pathway: PLD/LAH → β-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.

Figure 4.

Figure 4

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Time-course experiments of the activity of β-oxidation (○) and isocitrate lyase (•). Palmitoyl-CoA-dependent NADH production was used for β-oxidation activity. Extracts from cells harvested daily after starvation were used for the assays. Bar represents ± se of three replicates. U, Units.

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).

Figure 5.

Figure 5

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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 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.

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 β-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 γ-irradiated cauliflower microsomal membranes, PLD and LAH, but not PLC, were associated with the membrane degradation, and PLD activity was primarily induced upon γ-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.

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.

Abbreviations:

ICL

isocitrate lyase

LAH

lipolytic acyl hydrolase

PA

phosphatidic acid

PC

phosphatidylcholine

PLC

phospholipase C

PLD

phospholipase D

PtdBut

phosphatidyl butanol

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.

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