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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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Effect of treatments with ROS and NO scavengers on the percentage of microspore embryogenesis. (A) Control microspore culture at 20 days after the inductor stress treatment, micrograph showing large developing embryos (arrowheads), dense and large multicellular microspores (arrows), and small non-embryogenic microspores; bar, 50 μm. (B) Microspore culture treated with the NO scavenger, cPTIO, for 20 days after the inductor stress treatment, exhibiting some dense and large multicellular microspores (arrow) and many small non-embryogenic microspores; bar, 50 μm. (C) Histogram indicating the percentage of embryogenic structures (large embryos and multicellular microspores) in microspore cultures 20 days after the inductor stress treatment, without scavengers (control) or with treatments with Cl2Mn (O2.− scavenger), ascorbate (Asc, H2O2 scavenger), or cPTIO (NO scavenger). Different 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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fig9: Effect of treatments with ROS and NO scavengers on the percentage of microspore embryogenesis. (A) Control microspore culture at 20 days after the inductor stress treatment, micrograph showing large developing embryos (arrowheads), dense and large multicellular microspores (arrows), and small non-embryogenic microspores; bar, 50 μm. (B) Microspore culture treated with the NO scavenger, cPTIO, for 20 days after the inductor stress treatment, exhibiting some dense and large multicellular microspores (arrow) and many small non-embryogenic microspores; bar, 50 μm. (C) Histogram indicating the percentage of embryogenic structures (large embryos and multicellular microspores) in microspore cultures 20 days after the inductor stress treatment, without scavengers (control) or with treatments with Cl2Mn (O2.− scavenger), ascorbate (Asc, H2O2 scavenger), or cPTIO (NO scavenger). Different 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: To evaluate the elimination effect of ROS and NO on microspore cultures at later stages, treatments with specific scavengers, Cl2Mn (O2.− scavenger), ascorbate (H2O2 scavenger), and cPTIO (NO scavenger) were carried out on stress-treated microspore cultures. Treated and control microspore cultures were observed after 20 days and embryogenesis progression was analysed. The percentage of embryogenesis was calculated by quantification of non-responding microspores and developing embryo structures. In control microspore cultures, multicellular microspores of different sizes and larger developing embryos were observed (Fig. 9A), as well as smaller non-embryogenic microspores. However, in microspore cultures treated with the scavengers, no large embryos were observed in the culture at the same stage, only some embryogenic and multicellular microspores characterized by their large size and density, in comparison with the small non-embryogenic microspores (Fig. 9B). This indicated that the elimination of ROS and NO produced a delay in microspore-derived embryo development. The quantification of the embryogenic and non-embryogenic structures found in control and treated cultures showed significant percentage decreases of embryogenesis in the three cultures treated with the scavengers when compared with control cultures (Fig. 9C), indicating that scavenging of ROS and NO negatively affected microspore embryogenesis progression.


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)

Effect of treatments with ROS and NO scavengers on the percentage of microspore embryogenesis. (A) Control microspore culture at 20 days after the inductor stress treatment, micrograph showing large developing embryos (arrowheads), dense and large multicellular microspores (arrows), and small non-embryogenic microspores; bar, 50 μm. (B) Microspore culture treated with the NO scavenger, cPTIO, for 20 days after the inductor stress treatment, exhibiting some dense and large multicellular microspores (arrow) and many small non-embryogenic microspores; bar, 50 μm. (C) Histogram indicating the percentage of embryogenic structures (large embryos and multicellular microspores) in microspore cultures 20 days after the inductor stress treatment, without scavengers (control) or with treatments with Cl2Mn (O2.− scavenger), ascorbate (Asc, H2O2 scavenger), or cPTIO (NO scavenger). Different 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

fig9: Effect of treatments with ROS and NO scavengers on the percentage of microspore embryogenesis. (A) Control microspore culture at 20 days after the inductor stress treatment, micrograph showing large developing embryos (arrowheads), dense and large multicellular microspores (arrows), and small non-embryogenic microspores; bar, 50 μm. (B) Microspore culture treated with the NO scavenger, cPTIO, for 20 days after the inductor stress treatment, exhibiting some dense and large multicellular microspores (arrow) and many small non-embryogenic microspores; bar, 50 μm. (C) Histogram indicating the percentage of embryogenic structures (large embryos and multicellular microspores) in microspore cultures 20 days after the inductor stress treatment, without scavengers (control) or with treatments with Cl2Mn (O2.− scavenger), ascorbate (Asc, H2O2 scavenger), or cPTIO (NO scavenger). Different 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: To evaluate the elimination effect of ROS and NO on microspore cultures at later stages, treatments with specific scavengers, Cl2Mn (O2.− scavenger), ascorbate (H2O2 scavenger), and cPTIO (NO scavenger) were carried out on stress-treated microspore cultures. Treated and control microspore cultures were observed after 20 days and embryogenesis progression was analysed. The percentage of embryogenesis was calculated by quantification of non-responding microspores and developing embryo structures. In control microspore cultures, multicellular microspores of different sizes and larger developing embryos were observed (Fig. 9A), as well as smaller non-embryogenic microspores. However, in microspore cultures treated with the scavengers, no large embryos were observed in the culture at the same stage, only some embryogenic and multicellular microspores characterized by their large size and density, in comparison with the small non-embryogenic microspores (Fig. 9B). This indicated that the elimination of ROS and NO produced a delay in microspore-derived embryo development. The quantification of the embryogenic and non-embryogenic structures found in control and treated cultures showed significant percentage decreases of embryogenesis in the three cultures treated with the scavengers when compared with control cultures (Fig. 9C), indicating that scavenging of ROS and NO negatively affected microspore embryogenesis progression.

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