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Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures.

Doyle SM, Diamond M, McCabe PF - J. Exp. Bot. (2009)

Bottom Line: Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation.Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures.The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland.

ABSTRACT
Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 degrees C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

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Light-grown and dark-grown culture cells react differently to an AL-PCD-inducing heat treatment. Percentage of AL-PCD, necrosis, and living cells, scored using both cell morphology and DNA fragmentation markers of AL-PCD, in A. thaliana dark-grown and light-grown cultures, 24 h after heat treatment. Values are means ±SD (n ≥3 replicates). PCD values marked with an asterisk were significantly different from PCD in light-grown cultures for the same PCD marker (P=0.006 for cell morphology marker and P=0.001 for DNA fragmentation marker).
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fig3: Light-grown and dark-grown culture cells react differently to an AL-PCD-inducing heat treatment. Percentage of AL-PCD, necrosis, and living cells, scored using both cell morphology and DNA fragmentation markers of AL-PCD, in A. thaliana dark-grown and light-grown cultures, 24 h after heat treatment. Values are means ±SD (n ≥3 replicates). PCD values marked with an asterisk were significantly different from PCD in light-grown cultures for the same PCD marker (P=0.006 for cell morphology marker and P=0.001 for DNA fragmentation marker).

Mentions: A heat treatment of 10 min duration at 55 °C will induce AL-PCD in the majority of cells in plant suspension cultures (McCabe et al., 1997), including A. thaliana cultures. However, the level of AL-PCD induced by this treatment was significantly higher for dark-grown than for light-grown cultures and this was shown using both the cell morphology and DNA fragmentation markers of AL-PCD (Fig. 3). AL-PCD was measured 24 h after the heat treatment because it was found that maximal cell death and cell condensation levels were observable at this time (data not shown). According to the cell morphology marker, the fraction of AL-PCD induced by the heat treatment was 80% in dark-grown and 63% in light-grown cultures, with all remaining cells necrotic. Using the DNA fragmentation marker (TUNEL assay), the fraction of cells with nuclei positive for DNA fragmentation was 89% in dark-grown and 33% in light-grown cultures.


Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures.

Doyle SM, Diamond M, McCabe PF - J. Exp. Bot. (2009)

Light-grown and dark-grown culture cells react differently to an AL-PCD-inducing heat treatment. Percentage of AL-PCD, necrosis, and living cells, scored using both cell morphology and DNA fragmentation markers of AL-PCD, in A. thaliana dark-grown and light-grown cultures, 24 h after heat treatment. Values are means ±SD (n ≥3 replicates). PCD values marked with an asterisk were significantly different from PCD in light-grown cultures for the same PCD marker (P=0.006 for cell morphology marker and P=0.001 for DNA fragmentation marker).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803215&req=5

fig3: Light-grown and dark-grown culture cells react differently to an AL-PCD-inducing heat treatment. Percentage of AL-PCD, necrosis, and living cells, scored using both cell morphology and DNA fragmentation markers of AL-PCD, in A. thaliana dark-grown and light-grown cultures, 24 h after heat treatment. Values are means ±SD (n ≥3 replicates). PCD values marked with an asterisk were significantly different from PCD in light-grown cultures for the same PCD marker (P=0.006 for cell morphology marker and P=0.001 for DNA fragmentation marker).
Mentions: A heat treatment of 10 min duration at 55 °C will induce AL-PCD in the majority of cells in plant suspension cultures (McCabe et al., 1997), including A. thaliana cultures. However, the level of AL-PCD induced by this treatment was significantly higher for dark-grown than for light-grown cultures and this was shown using both the cell morphology and DNA fragmentation markers of AL-PCD (Fig. 3). AL-PCD was measured 24 h after the heat treatment because it was found that maximal cell death and cell condensation levels were observable at this time (data not shown). According to the cell morphology marker, the fraction of AL-PCD induced by the heat treatment was 80% in dark-grown and 63% in light-grown cultures, with all remaining cells necrotic. Using the DNA fragmentation marker (TUNEL assay), the fraction of cells with nuclei positive for DNA fragmentation was 89% in dark-grown and 33% in light-grown cultures.

Bottom Line: Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation.Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures.The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland.

ABSTRACT
Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 degrees C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

Show MeSH
Related in: MedlinePlus