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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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Stress-induced embryogenesis in isolated barley microspore cultures and plantlet regeneration. (A) Isolated microspores at culture initiation. (B) Microspore-derived embryos after 28 days in culture. (C) Germination of microspore-derived embryos. (D) Regenerated plantlet in solid MS medium. Bars, 0.2 mm (A), 10 mm (B–D). (This figure is available in colour at JXB online.)
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fig2: Stress-induced embryogenesis in isolated barley microspore cultures and plantlet regeneration. (A) Isolated microspores at culture initiation. (B) Microspore-derived embryos after 28 days in culture. (C) Germination of microspore-derived embryos. (D) Regenerated plantlet in solid MS medium. Bars, 0.2 mm (A), 10 mm (B–D). (This figure is available in colour at JXB online.)

Mentions: Stress treatment of in vitro cultures of isolated microspores (Fig. 2A) can induce them to generate embryos that germinate and produce haploid and double-haploid plants. Four days after the cold stress treatment, cultures yielded multi-nuclear structures or pro-embryos which proceeded to develop into embryos in the following 28 days (Fig. 2B–D). Later, 45–53-day-old embryos induced on KBP media were cultured for germination on MS medium containing 2% sucrose, gelled with 0.7% agar, and with a pH of 5.8. The embryos which were maintained at 25 °C, first in the dark for 4 days and then in the light (Fig. 2C), finally regenerated green plantlets (Fig. 2D), around 50% of which were diploid, as assessed by flow cytometry using propidium iodide staining of isolated nuclei (Wilkinson et al., 2000). To investigate the developmental pathways by which cold treatment induces microspore embryogenesis in barley, the present study studied the number and morphology of nuclei using DNA condensation as a marker to distinguish cells with vegetative and generative origins (Sunderland et al., 1979; Testillano et al., 2004) by analysing squash preparations of microspores in cultures stained with 4′,6-diamidino-2-phenylindole under fluorescence and phase contrast microscopy, as well as by staining semi-thin sections with toluidine blue. The spikes selected to start the cultures mostly contained vacuolated microspores which are characterized by a large vacuole (Fig. 3A,E,I). After the cold stress pre-treatment (spikes kept for 23 days at 4 °C) to induce embryogenesis, the microspores reprogrammed and started a new pathway. During the cold treatment, the first mitotic division produced two equal nuclei. After 4 days in culture (Fig. 3B,F,J) pro-embryos with up to 10 nuclei could be observed. Later, subsequent divisions produced multicellular embryos with exine (Fig. 3C,G,K) which finally broke, yielding the embryos (Fig. 3D,H,L) which would give rise to haploid and double-haploid plants.


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)

Stress-induced embryogenesis in isolated barley microspore cultures and plantlet regeneration. (A) Isolated microspores at culture initiation. (B) Microspore-derived embryos after 28 days in culture. (C) Germination of microspore-derived embryos. (D) Regenerated plantlet in solid MS medium. Bars, 0.2 mm (A), 10 mm (B–D). (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

fig2: Stress-induced embryogenesis in isolated barley microspore cultures and plantlet regeneration. (A) Isolated microspores at culture initiation. (B) Microspore-derived embryos after 28 days in culture. (C) Germination of microspore-derived embryos. (D) Regenerated plantlet in solid MS medium. Bars, 0.2 mm (A), 10 mm (B–D). (This figure is available in colour at JXB online.)
Mentions: Stress treatment of in vitro cultures of isolated microspores (Fig. 2A) can induce them to generate embryos that germinate and produce haploid and double-haploid plants. Four days after the cold stress treatment, cultures yielded multi-nuclear structures or pro-embryos which proceeded to develop into embryos in the following 28 days (Fig. 2B–D). Later, 45–53-day-old embryos induced on KBP media were cultured for germination on MS medium containing 2% sucrose, gelled with 0.7% agar, and with a pH of 5.8. The embryos which were maintained at 25 °C, first in the dark for 4 days and then in the light (Fig. 2C), finally regenerated green plantlets (Fig. 2D), around 50% of which were diploid, as assessed by flow cytometry using propidium iodide staining of isolated nuclei (Wilkinson et al., 2000). To investigate the developmental pathways by which cold treatment induces microspore embryogenesis in barley, the present study studied the number and morphology of nuclei using DNA condensation as a marker to distinguish cells with vegetative and generative origins (Sunderland et al., 1979; Testillano et al., 2004) by analysing squash preparations of microspores in cultures stained with 4′,6-diamidino-2-phenylindole under fluorescence and phase contrast microscopy, as well as by staining semi-thin sections with toluidine blue. The spikes selected to start the cultures mostly contained vacuolated microspores which are characterized by a large vacuole (Fig. 3A,E,I). After the cold stress pre-treatment (spikes kept for 23 days at 4 °C) to induce embryogenesis, the microspores reprogrammed and started a new pathway. During the cold treatment, the first mitotic division produced two equal nuclei. After 4 days in culture (Fig. 3B,F,J) pro-embryos with up to 10 nuclei could be observed. Later, subsequent divisions produced multicellular embryos with exine (Fig. 3C,G,K) which finally broke, yielding the embryos (Fig. 3D,H,L) which would give rise to haploid and double-haploid plants.

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