Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus

Abstract

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Keywords: Autophagy; Metacaspase; Microspore embryogenesis; Programmed cell death; Rapeseed; Stress.

Fig. 1

Stress-induced microspore embryogenesis in B. napus. Micrographs of toluidine blue-stained sections of (A) isolated vacuolated microspore, before stress (B) proembryo of stress-treated microspore culture, (C) globular embryo and (D) cotyledonary embryo. (E) Petry dish containing microspore-derived embryos and (F) higher magnification of cotyledonary embryos. Ex: exine; N: nucleus; V: vacuole. Scale bars: (A–C): 10 �m; (D): 500 �m; (E): 10 mm; and (F): 1 mm.

Fig. 2

Cell death in stress-induced microspore embryogenesis. Representative micrographs of Evan’s blue staining showing cell death in: (A) microspores (m) before stress; (B) stress-treated microspore culture, containing microspores (m) and proembryos (p); (C) 10-day culture composed by globular embryos (e), as well as microspores (m) and proembryos (p). Arrows: dead microspores; arrowheads: dead proembryos; Scale bars: 10 �m. (D) Quantification of cell death. The data represent mean � SEM. Different letters indicate significant differences (chi-square test at P < 0.05).

Fig. 3

MCA activity in microspore embryogenesis. Effect of pH (A), EDTA (B) and protease inhibitors at 10 �M (C) on VRPRase activity of protein extracts from microspore-derived embryos and recombinant mcII-Pa. The data represent mean � SEM. Different letters indicate significant differences within protein extracts or recombinant mcII-Pa series (Tukey’s t-test at P < 0.05). Quantification of cell death in control and Z-VRPR-FMK-treated microspore cultures, at two stages: stress-treated microspore culture (D) and 10-day culture (E). Different letters indicate significant differences (chi-square test at P < 0.05).

Fig. 4

MCA protein family of B. napus and expression of selected genes during microspore embryogenesis. (A) Phylogenetic tree of Arabidopsis and B. napus MCAs inferred using the maximum-likelihood method with 1,000 bootstrap replicates using MEGA software. Percentages of taxa clustered together are shown next to the branches. The schematic domain structure of individual MCAs is shown to the right. (B) Relative changes in mRNA levels of BnMCA-Ia, BnMCA-IIa and BnMCA-IIi normalized to vacuolated microspore levels determined by RT-qPCR. The data represent mean � SEM. Different letters indicate significant differences (Student’s t-test at P < 0.05).

Fig. 5

Expression and localization of autophagy-related proteins in microspore embryogenesis. (A) BnATG5 mRNA levels in stress-treated microspores compared to vacuolated microspores before stress, as determined by RT-qPCR. The data represent mean � SEM. Expression levels were normalized to vacuolated microspores. Different letters indicate significant differences (Student’s t-test at P < 0.05). Immunofluorescence and confocal microscopy analysis of ATG5 (B) and ATG8 (C) in vacuolated microspores (before stress) and stress-treated microspores (after stress). Left panels are merged images of Normarsky’s differential interference contrast, ATG5 or ATG8 immunofluorescence signal (green) and DAPI staining of nuclei (blue). Right panels are merged images of ATG5 or ATG8 immunofluorescence signal (green) and DAPI staining of nuclei (blue). Scale bars: 10 �m.

Fig. 6

Electron microscopy analysis of autophagosomes and ATG8 localization in microspores. (A) Cytoplasmic area of vacuolated microspore, before stress. (B) Cytoplasmic area of stress-treated microspore treated with E-64d to preserve autophagic bodies in the vacuoles (arrows). (C) Autophagosome in stress-treated microspore. (D and E) Immunogold labeling of ATG8 (arrows) is restricted to autophagosomes in stress-treated microspores. V, vacuole; M, mitochondria; S, starch granule. Scale bars: (A, B) 1 �m; (C–E) 0.2 �m.

Fig. 7

Autophagic flux analysis and effect of autophagy and MCA inhibitors on cell death and microspore embryogenesis. (A) Immunoblot analysis of NBR1 degradation in stress-induced microspores, with and without E-64d treatment (7.5 �M). Ponceau staining of total proteins was used as a loading control. (B) Quantification of bands in A using ImageJ and two-step normalization: first, to control stress-treated microspore and then to corresponding intensities of Ponceau staining (loading). (C) RT-qPCR analysis of BnNBR1 mRNA levels. The values were normalized to control, stress-treated microspore samples. The data represent mean � SEM. Asterisks in (B) and (C) indicate significant differences (Student’s t-test at P < 0.05). (D) Effect of E-64d on cell death in stress-induced microspore cultures. (E) Effect of individual and combined treatments with E64d and Z-VRPR-FMK on cell death in stress-induced microspores. (F) Quantification of proembryos (a marker of embryogenesis initiation) after stress treatment in control cultures and cultures treated with E-64d and/or Z-VRPR-FMK. The data represent mean � SEM. Different letters in (D)–(F) indicate significant differences (chi-square test at P < 0.05).