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Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus

Eduardo Berenguer
Eduardo Berenguer
, 
María-Teresa Solís
María-Teresa Solís
, 
Yolanda Pérez-Pérez
Yolanda Pérez-Pérez
 and 
Pilar S. Testillano
Pilar S. Testillano
   | Jul 25, 2019
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Article Category: Research Article
Published Online: Jul 25, 2019
Page range: 152 - 159
DOI: https://doi.org/10.2478/ebtj-2019-0018
Keywords
© 2019 Eduardo Berenguer, María-Teresa Solís, Yolanda Pérez-Pérez, Pilar S. Testillano, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Figure 1

Main stages of stress-induced microspore embryogenesis in Brassica napus Micrographs of toluidine blue-stained sections, for cell structure analysis in (A) isolated vacuolated microspore at culture initiation and (B) proembryo, formed in microspore culture, 4 days after stress, still surrounded by the exine (Ex). (C) Panoramic view of microspore-derived cotyledonary embryos, in the Petri dish. (D) Higher magnification view, showing a detail of a fully differentiated cotyledonary embryo. V: vacuole; N: nucleus; Ex: exine. Scale bars: (A-B) 5 μm; (C) 1 cm; (D) 1 mm.
Main stages of stress-induced microspore embryogenesis in Brassica napus Micrographs of toluidine blue-stained sections, for cell structure analysis in (A) isolated vacuolated microspore at culture initiation and (B) proembryo, formed in microspore culture, 4 days after stress, still surrounded by the exine (Ex). (C) Panoramic view of microspore-derived cotyledonary embryos, in the Petri dish. (D) Higher magnification view, showing a detail of a fully differentiated cotyledonary embryo. V: vacuole; N: nucleus; Ex: exine. Scale bars: (A-B) 5 μm; (C) 1 cm; (D) 1 mm.

Figure 2

Detection of cell death in microspore culture, before stress. Representative micrograph of dead microspores revealed by Evans blue staining in isolated microspore culture, before the inductor stress. Arrows: dead cells. Scale bars: 10 μm.
Detection of cell death in microspore culture, before stress. Representative micrograph of dead microspores revealed by Evans blue staining in isolated microspore culture, before the inductor stress. Arrows: dead cells. Scale bars: 10 μm.

Figure 3

Cell death quantification in stress-induced microspore embryogenesis in B. napus Histogram showing the percentage of dead cells identified by Evans blue staining before and after stress treatment. Columns represent mean ± S.E.M; Asterisk indicate significant differences (Student’s t-test at p < 0.05).
Cell death quantification in stress-induced microspore embryogenesis in B. napus Histogram showing the percentage of dead cells identified by Evans blue staining before and after stress treatment. Columns represent mean ± S.E.M; Asterisk indicate significant differences (Student’s t-test at p < 0.05).

Figure 4

Caspase 3-like activity in microspore cultures in B. napus Histogram showing caspase 3-like activity (black columns) and negative controls with the caspase 3 inhibitor (grey columns) before and after stress treatment. Columns represent mean ± S.E.M. Asterisk indicate significant differences (Student’s t-test at p < 0.05).
Caspase 3-like activity in microspore cultures in B. napus Histogram showing caspase 3-like activity (black columns) and negative controls with the caspase 3 inhibitor (grey columns) before and after stress treatment. Columns represent mean ± S.E.M. Asterisk indicate significant differences (Student’s t-test at p < 0.05).

Figure 5

Quantification of cell death levels in control and Ac-DEVD-CHO-treated microspore cultures. Histogram showing the percentage of dead cells identified by Evans blue staining in control and Ac-DEVD-CHO-treated cultures, 4 days after stress. Columns represent mean ± S.E.M; Asterisk indicate significant differences (ANOVA and Tukey’s test at p < 0.05).
Quantification of cell death levels in control and Ac-DEVD-CHO-treated microspore cultures. Histogram showing the percentage of dead cells identified by Evans blue staining in control and Ac-DEVD-CHO-treated cultures, 4 days after stress. Columns represent mean ± S.E.M; Asterisk indicate significant differences (ANOVA and Tukey’s test at p < 0.05).

Figure 6

Effects of Ac-DEVD-CHO on microspore embryogenesis induction. Representative micrographs of microspore cultures showing proembryos formed (pointed by arrows) in (A) control cultures and (B) Ac-DEVD-CHO-treated cultures, 4 days after stress. Arrowhead: proembryo with the breakdown of exine wall. Scale bars: 50 μm.
Effects of Ac-DEVD-CHO on microspore embryogenesis induction. Representative micrographs of microspore cultures showing proembryos formed (pointed by arrows) in (A) control cultures and (B) Ac-DEVD-CHO-treated cultures, 4 days after stress. Arrowhead: proembryo with the breakdown of exine wall. Scale bars: 50 μm.

Figure 7

Quantification of embryogenesis induction levels in control and Ac-DEVD-CHO-treated microspore cultures. Histogram showing the percentage of proembryos formed in control and Ac-DEVD-CHO-treated cultures, 4 days after stress. Columns represent mean ± S.E.M; Asterisk indicate significant differences (ANOVA and Tukey’s test at p < 0.05).
Quantification of embryogenesis induction levels in control and Ac-DEVD-CHO-treated microspore cultures. Histogram showing the percentage of proembryos formed in control and Ac-DEVD-CHO-treated cultures, 4 days after stress. Columns represent mean ± S.E.M; Asterisk indicate significant differences (ANOVA and Tukey’s test at p < 0.05).
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