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Arabidopsis non-specific phospholipase C1: characterization and its involvement in response to heat stress

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ABSTRACT

The Arabidopsis non-specific phospholipase C1 (NPC) protein family is encoded by the genes NPC1 – NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat.

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Effect of heat stress on NPC activity in BY-2 tobacco cells. Six-day-old BY-2 cells were incubated with bodipy-PC as a substrate for 10 min. Half of the cells were transferred to 42°C, and the rest were kept at 20°C. The reaction was stopped at the times indicated. Lipids were extracted and separated by HP-TLC, and bodipy-DAG fluorescence was quantified. (A) HP-TLC chromatogram of bodipy-DAG, the product of NPC activity. (B) The quantification of bodipy-DAG fluorescence. Data represent means ± SD from independently analyzed parallel samples. This experiment was repeated twice with similar results. DAG, diacylglycerol; HP-TLC, high-performance thin layer chromatography; NPC, non-specific phospholipase C; PC, phosphatidylcholine.
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Figure 4: Effect of heat stress on NPC activity in BY-2 tobacco cells. Six-day-old BY-2 cells were incubated with bodipy-PC as a substrate for 10 min. Half of the cells were transferred to 42°C, and the rest were kept at 20°C. The reaction was stopped at the times indicated. Lipids were extracted and separated by HP-TLC, and bodipy-DAG fluorescence was quantified. (A) HP-TLC chromatogram of bodipy-DAG, the product of NPC activity. (B) The quantification of bodipy-DAG fluorescence. Data represent means ± SD from independently analyzed parallel samples. This experiment was repeated twice with similar results. DAG, diacylglycerol; HP-TLC, high-performance thin layer chromatography; NPC, non-specific phospholipase C; PC, phosphatidylcholine.

Mentions: Several studies revealed that phospholipases, namely PI-PLC and PLD, play a role in HS (Mishkind et al., 2009; Zheng et al., 2012; Gao et al., 2014). To test wheteher NPC1 may be also involved in HS, we first measured NPC activity in BY-2 cells using a fluorescently labelled substrate (Pejchar et al., 2010, 2013) to determine whether activity changes after HS. We incubated bodipy-PC with BY-2 cells for 10 min. We then transferred half of the cells to 42°C; the remaining cells were kept under control conditions (20°C). The reaction was stopped immediately (0 min) or after 5, 15, or 30 min. Lipids were extracted and separated by HP-TLC. Spots corresponding to bodipy-DAG were quantified (Figure 4). The level of bodipy-DAG in this experimental setup corresponds mainly to NPC activity (Pejchar et al., 2010). However, the assay cannot fully exclude the possibility that PLD/PAP pathway is partially involved. We found increasing, time-dependent NPC activity during HS. The NPC activity increased fourfold in stressed cells compared with the control after 5 min; NPC activity was six- and ninefold greater after 15 and 30 min, respectively (Figure 4). We also measured activity during recovery after HS. The cells were stressed for 30 min and then returned to control conditions. Activity was measured after 15 min, 1, 3, 5, and 24 h of recovery. We observed that the activity remained higher in stressed cells in the first hour after HS. Later, the NPC activity in stressed cells returned to the level of control cells (data not shown). Taken together, these results suggest that NPC activity increased rapidly during HS and stays high during the short, transient period of recovery after HS in BY-2 cells.


Arabidopsis non-specific phospholipase C1: characterization and its involvement in response to heat stress
Effect of heat stress on NPC activity in BY-2 tobacco cells. Six-day-old BY-2 cells were incubated with bodipy-PC as a substrate for 10 min. Half of the cells were transferred to 42°C, and the rest were kept at 20°C. The reaction was stopped at the times indicated. Lipids were extracted and separated by HP-TLC, and bodipy-DAG fluorescence was quantified. (A) HP-TLC chromatogram of bodipy-DAG, the product of NPC activity. (B) The quantification of bodipy-DAG fluorescence. Data represent means ± SD from independently analyzed parallel samples. This experiment was repeated twice with similar results. DAG, diacylglycerol; HP-TLC, high-performance thin layer chromatography; NPC, non-specific phospholipase C; PC, phosphatidylcholine.
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Related In: Results  -  Collection

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Figure 4: Effect of heat stress on NPC activity in BY-2 tobacco cells. Six-day-old BY-2 cells were incubated with bodipy-PC as a substrate for 10 min. Half of the cells were transferred to 42°C, and the rest were kept at 20°C. The reaction was stopped at the times indicated. Lipids were extracted and separated by HP-TLC, and bodipy-DAG fluorescence was quantified. (A) HP-TLC chromatogram of bodipy-DAG, the product of NPC activity. (B) The quantification of bodipy-DAG fluorescence. Data represent means ± SD from independently analyzed parallel samples. This experiment was repeated twice with similar results. DAG, diacylglycerol; HP-TLC, high-performance thin layer chromatography; NPC, non-specific phospholipase C; PC, phosphatidylcholine.
Mentions: Several studies revealed that phospholipases, namely PI-PLC and PLD, play a role in HS (Mishkind et al., 2009; Zheng et al., 2012; Gao et al., 2014). To test wheteher NPC1 may be also involved in HS, we first measured NPC activity in BY-2 cells using a fluorescently labelled substrate (Pejchar et al., 2010, 2013) to determine whether activity changes after HS. We incubated bodipy-PC with BY-2 cells for 10 min. We then transferred half of the cells to 42°C; the remaining cells were kept under control conditions (20°C). The reaction was stopped immediately (0 min) or after 5, 15, or 30 min. Lipids were extracted and separated by HP-TLC. Spots corresponding to bodipy-DAG were quantified (Figure 4). The level of bodipy-DAG in this experimental setup corresponds mainly to NPC activity (Pejchar et al., 2010). However, the assay cannot fully exclude the possibility that PLD/PAP pathway is partially involved. We found increasing, time-dependent NPC activity during HS. The NPC activity increased fourfold in stressed cells compared with the control after 5 min; NPC activity was six- and ninefold greater after 15 and 30 min, respectively (Figure 4). We also measured activity during recovery after HS. The cells were stressed for 30 min and then returned to control conditions. Activity was measured after 15 min, 1, 3, 5, and 24 h of recovery. We observed that the activity remained higher in stressed cells in the first hour after HS. Later, the NPC activity in stressed cells returned to the level of control cells (data not shown). Taken together, these results suggest that NPC activity increased rapidly during HS and stays high during the short, transient period of recovery after HS in BY-2 cells.

View Article: PubMed Central

ABSTRACT

The Arabidopsis non-specific phospholipase C1 (NPC) protein family is encoded by the genes NPC1 – NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat.

No MeSH data available.


Related in: MedlinePlus