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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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Morphology of living and AL-PCD cells from A. thaliana cell suspension cultures. (A, B) Living cells from (A) light-grown culture and (B) dark-grown culture. Note the large organelles in the light-grown cells (A), indicated by the two arrows, which were absent from the dark-grown cells (B). (C, D) Dead cells from (C) light-grown culture and (D) dark-grown culture, 24 h after heat treatment. These cells displayed the AL-PCD hallmark morphology of cell condensation, visible as a gap between the cell wall and cytoplasm, indicated by the arrows in both (C) and (D). Note that the cell condensation was far more extreme in dark-grown (D) than in light-grown (C) cells. Scale bars represent 20 μm.
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fig1: Morphology of living and AL-PCD cells from A. thaliana cell suspension cultures. (A, B) Living cells from (A) light-grown culture and (B) dark-grown culture. Note the large organelles in the light-grown cells (A), indicated by the two arrows, which were absent from the dark-grown cells (B). (C, D) Dead cells from (C) light-grown culture and (D) dark-grown culture, 24 h after heat treatment. These cells displayed the AL-PCD hallmark morphology of cell condensation, visible as a gap between the cell wall and cytoplasm, indicated by the arrows in both (C) and (D). Note that the cell condensation was far more extreme in dark-grown (D) than in light-grown (C) cells. Scale bars represent 20 μm.

Mentions: Using fluorescein diacetate as a vital stain, cells were scored as either alive, dead via necrosis, or dead via AL-PCD with the light microscope, using the methods described by McCabe and Leaver (2000) and Reape et al. (2008). Dead cells that displayed the hallmark morphology of condensed cell, retracted away from the cell wall, were scored as dead via AL-PCD (see Fig. 1). Dead cells that were not condensed were scored as necrotic. Viable cells were not condensed. AL-PCD can also be scored by observing the hallmark feature of DNA fragmentation (McCabe et al., 1997; McCabe and Leaver, 2000). The fluorescent FragEL kit (Calbiochem) was used to perform the TUNEL (TdT-mediated deoxyuridine triphosphate nick end labelling) assay to identify DNA fragmentation. Samples were maintained at 4 °C during the procedure and washed with TBS between each step. Cells were fixed in 4% w/v formaldehyde in PBS for 30 min, before being permeabilized in 20 μg ml−1 proteinase K in 10 mM TRIS, pH 8, at room temperature for 5 min. Cells were then transferred to equilibration buffer at room temperature for 20 min and then incubated in a prepared mixture of TdT enzyme and labelling reaction mix at 37 °C in darkness for 1.5 h for fragment end labelling. Cells were then mounted in DAPI and DAPI-stained nuclei and fluorescein-labelled nuclei, containing fragmented DNA, were observed with fluorescence microscopy.


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)

Morphology of living and AL-PCD cells from A. thaliana cell suspension cultures. (A, B) Living cells from (A) light-grown culture and (B) dark-grown culture. Note the large organelles in the light-grown cells (A), indicated by the two arrows, which were absent from the dark-grown cells (B). (C, D) Dead cells from (C) light-grown culture and (D) dark-grown culture, 24 h after heat treatment. These cells displayed the AL-PCD hallmark morphology of cell condensation, visible as a gap between the cell wall and cytoplasm, indicated by the arrows in both (C) and (D). Note that the cell condensation was far more extreme in dark-grown (D) than in light-grown (C) cells. Scale bars represent 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Morphology of living and AL-PCD cells from A. thaliana cell suspension cultures. (A, B) Living cells from (A) light-grown culture and (B) dark-grown culture. Note the large organelles in the light-grown cells (A), indicated by the two arrows, which were absent from the dark-grown cells (B). (C, D) Dead cells from (C) light-grown culture and (D) dark-grown culture, 24 h after heat treatment. These cells displayed the AL-PCD hallmark morphology of cell condensation, visible as a gap between the cell wall and cytoplasm, indicated by the arrows in both (C) and (D). Note that the cell condensation was far more extreme in dark-grown (D) than in light-grown (C) cells. Scale bars represent 20 μm.
Mentions: Using fluorescein diacetate as a vital stain, cells were scored as either alive, dead via necrosis, or dead via AL-PCD with the light microscope, using the methods described by McCabe and Leaver (2000) and Reape et al. (2008). Dead cells that displayed the hallmark morphology of condensed cell, retracted away from the cell wall, were scored as dead via AL-PCD (see Fig. 1). Dead cells that were not condensed were scored as necrotic. Viable cells were not condensed. AL-PCD can also be scored by observing the hallmark feature of DNA fragmentation (McCabe et al., 1997; McCabe and Leaver, 2000). The fluorescent FragEL kit (Calbiochem) was used to perform the TUNEL (TdT-mediated deoxyuridine triphosphate nick end labelling) assay to identify DNA fragmentation. Samples were maintained at 4 °C during the procedure and washed with TBS between each step. Cells were fixed in 4% w/v formaldehyde in PBS for 30 min, before being permeabilized in 20 μg ml−1 proteinase K in 10 mM TRIS, pH 8, at room temperature for 5 min. Cells were then transferred to equilibration buffer at room temperature for 20 min and then incubated in a prepared mixture of TdT enzyme and labelling reaction mix at 37 °C in darkness for 1.5 h for fragment end labelling. Cells were then mounted in DAPI and DAPI-stained nuclei and fluorescein-labelled nuclei, containing fragmented DNA, were observed with fluorescence microscopy.

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