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NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

Rodríguez-Serrano M, Bárány I, Prem D, Coronado MJ, Risueño MC, Testillano PS - J. Exp. Bot. (2011)

Bottom Line: Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages.In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions.Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores.

View Article: PubMed Central - PubMed

Affiliation: Plant Development and Nuclear Architecture, Centro de Investigaciones Biológicas, Madrid, Spain.

ABSTRACT
Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

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Cell death and caspase 3-like activity in barley embryogenic suspension cultures. (A) Cells of suspension culture visualized by differential interference contrast microscopy (upper) and 4′,6-diamidino-2-phenylindole staining for DNA revealing the nuclei (lower); bar, 45 μm. (B) Histogram showing the percentage of dead cells identified by Evans Blue staining in control and stress-treated cells. (C) Evans Blue staining revealing dead cells in blue (arrow) and living cells unstained (arrowhead); bar, 45 μm. (D) Caspase 3-like activity in control and stress-treated cells, as well as in stress-treated cells with the caspase 3 inhibitor as control. Letters indicate significant differences at P < 0.05 according to Duncan’s multiple-range test (This figure is available in colour at JXB online.)
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fig11: Cell death and caspase 3-like activity in barley embryogenic suspension cultures. (A) Cells of suspension culture visualized by differential interference contrast microscopy (upper) and 4′,6-diamidino-2-phenylindole staining for DNA revealing the nuclei (lower); bar, 45 μm. (B) Histogram showing the percentage of dead cells identified by Evans Blue staining in control and stress-treated cells. (C) Evans Blue staining revealing dead cells in blue (arrow) and living cells unstained (arrowhead); bar, 45 μm. (D) Caspase 3-like activity in control and stress-treated cells, as well as in stress-treated cells with the caspase 3 inhibitor as control. Letters indicate significant differences at P < 0.05 according to Duncan’s multiple-range test (This figure is available in colour at JXB online.)

Mentions: The embryogenic suspension cultures consisted of elongated cells exhibiting a small nucleus and large cytoplasm with big vacuoles (Fig. 11A). The study analysed the effects of the stress treatments used for embryogenesis induction on barley microspore cultures, such as cold stress or starvation, focusing specifically on cell death occurrence and oxidative and nitrosative damage. Application of cold stress (4 °C) to embryogenic suspension cultures resulted in the complete cell death of the culture (data not shown); for this reason, the other stress condition that also induces embryogenesis in barley microspore cultures, namely pre-culture in a starvation medium, was used (Joähne-Gärtner and Lörz, 1999; Coronado et al., 2005). The quantification of the results and the microscopic images of Evans Blue staining (Fig. 11B,C) showed low cell death levels in control cells, whereas the proportion of dead cells increased approximately 2-fold after stress treatment (stress-treated cells) (Fig. 11B). In addition, caspase 3 activity increased 4-fold in the embryogenic suspension cultures after stress treatment (Fig. 11D). The increase in cell death was accompanied by an increase in caspase 3-like activity in both culture systems, while treatments with specific caspase 3 inhibitors diminished cell death proportions. The effects of stress on increases in cell death levels and caspase 3 activity in suspension cultures were similar to those obtained in microspore cultures and suggested that PCD played an important role in the stress response of both systems.


NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

Rodríguez-Serrano M, Bárány I, Prem D, Coronado MJ, Risueño MC, Testillano PS - J. Exp. Bot. (2011)

Cell death and caspase 3-like activity in barley embryogenic suspension cultures. (A) Cells of suspension culture visualized by differential interference contrast microscopy (upper) and 4′,6-diamidino-2-phenylindole staining for DNA revealing the nuclei (lower); bar, 45 μm. (B) Histogram showing the percentage of dead cells identified by Evans Blue staining in control and stress-treated cells. (C) Evans Blue staining revealing dead cells in blue (arrow) and living cells unstained (arrowhead); bar, 45 μm. (D) Caspase 3-like activity in control and stress-treated cells, as well as in stress-treated cells with the caspase 3 inhibitor as control. Letters indicate significant differences at P < 0.05 according to Duncan’s multiple-range test (This figure is available in colour at JXB online.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3295391&req=5

fig11: Cell death and caspase 3-like activity in barley embryogenic suspension cultures. (A) Cells of suspension culture visualized by differential interference contrast microscopy (upper) and 4′,6-diamidino-2-phenylindole staining for DNA revealing the nuclei (lower); bar, 45 μm. (B) Histogram showing the percentage of dead cells identified by Evans Blue staining in control and stress-treated cells. (C) Evans Blue staining revealing dead cells in blue (arrow) and living cells unstained (arrowhead); bar, 45 μm. (D) Caspase 3-like activity in control and stress-treated cells, as well as in stress-treated cells with the caspase 3 inhibitor as control. Letters indicate significant differences at P < 0.05 according to Duncan’s multiple-range test (This figure is available in colour at JXB online.)
Mentions: The embryogenic suspension cultures consisted of elongated cells exhibiting a small nucleus and large cytoplasm with big vacuoles (Fig. 11A). The study analysed the effects of the stress treatments used for embryogenesis induction on barley microspore cultures, such as cold stress or starvation, focusing specifically on cell death occurrence and oxidative and nitrosative damage. Application of cold stress (4 °C) to embryogenic suspension cultures resulted in the complete cell death of the culture (data not shown); for this reason, the other stress condition that also induces embryogenesis in barley microspore cultures, namely pre-culture in a starvation medium, was used (Joähne-Gärtner and Lörz, 1999; Coronado et al., 2005). The quantification of the results and the microscopic images of Evans Blue staining (Fig. 11B,C) showed low cell death levels in control cells, whereas the proportion of dead cells increased approximately 2-fold after stress treatment (stress-treated cells) (Fig. 11B). In addition, caspase 3 activity increased 4-fold in the embryogenic suspension cultures after stress treatment (Fig. 11D). The increase in cell death was accompanied by an increase in caspase 3-like activity in both culture systems, while treatments with specific caspase 3 inhibitors diminished cell death proportions. The effects of stress on increases in cell death levels and caspase 3 activity in suspension cultures were similar to those obtained in microspore cultures and suggested that PCD played an important role in the stress response of both systems.

Bottom Line: Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages.In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions.Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores.

View Article: PubMed Central - PubMed

Affiliation: Plant Development and Nuclear Architecture, Centro de Investigaciones Biológicas, Madrid, Spain.

ABSTRACT
Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

Show MeSH
Related in: MedlinePlus