Figure 4. Deficiency of either autophagy or metacaspase switches the mode of cell death from vacuolar to necrotic. (A–C) Cell morphology in control and RNAi lines (exemplified by an ATG6 RNAi line). N, nucleus; V, vacuole; asterisks, cell wall. (A) A meristematic cell from the EM of the control line. (B) Vacuolated suspensor cell with intact plasma membrane and turgid protoplast. (C) Dead cells from an RNAi line with shrunken protoplasts (double arrows denote detachment of plasma membranes from the cell walls) and ruptured plasma membranes (inset, arrow). (D) Frequency of cells with compromised plasma membranes assessed by SYTOX orange staining of meristematic (M; control line), suspensor (S; control line), and necrotic (N; RNAi line) cells. Data represent the means ± SEM from assessing all cells in ≥10 embryogenic structures per line. (E–G) Mitochondria in control and RNAi lines (exemplified by an ATG6 RNAi line). (E) Number of intact mitochondria per micrometer squared of the cytoplasm counted on the micrographs of meristematic, suspensor, and necrotic cells. Data represent the means ± SEM from three independent experiments, each including at least four cells per cell type. (F) Intact mitochondria (arrows) in a suspensor cell from the control line. (G) Swollen and degraded mitochondria (arrows) in a cell from an RNAi line. (H and I) Intracellular ATP content (H) and oxygen consumption (I). Data represent the means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01 (vs. control, Dunnett’s test). FW, fresh weight. (J and K) Cell vacuolization (J) and number of autophagosomes in the cytoplasm (K) estimated from the micrographs of meristematic (control line), suspensor (control line), and necrotic (ATG6 RNAi line) cells. Data represent the means ± SEM from three independent experiments, each including at least four cells per cell type. (L) Autophagosomes (open arrows) in the cytoplasm and autophagic bodies (closed arrows) in the vacuole of the embryo suspensor cell from a control line. (M and N) Quantification of necrotic death in control and RNAi lines after staining with FDA and FM4-64. (M) Staining patterns of EM and suspensor cells (control line) and necrotic cells (RNAi lines). DIC, differential interference contrast microscopy. (N) Frequency of necrotic cells. Data represent the means ± SEM from two independent experiments, each including two lines per genotype and ≥50 cells per line (P < 0.001; vs. control, Dunnett’s test). Different letters in D, E, J, and K indicate statistically different means (P < 0.001; Student’s t test). Bars: (A–C, main image) 5 µm; (C, inset) 0.5 µm; (F, G, and L) 2 µm; (M) 10 µm.
Figure 4.

Deficiency of either autophagy or metacaspase switches the mode of cell death from vacuolar to necrotic. (A–C) Cell morphology in control and RNAi lines (exemplified by an ATG6 RNAi line). N, nucleus; V, vacuole; asterisks, cell wall. (A) A meristematic cell from the EM of the control line. (B) Vacuolated suspensor cell with intact plasma membrane and turgid protoplast. (C) Dead cells from an RNAi line with shrunken protoplasts (double arrows denote detachment of plasma membranes from the cell walls) and ruptured plasma membranes (inset, arrow). (D) Frequency of cells with compromised plasma membranes assessed by SYTOX orange staining of meristematic (M; control line), suspensor (S; control line), and necrotic (N; RNAi line) cells. Data represent the means ± SEM from assessing all cells in ≥10 embryogenic structures per line. (E–G) Mitochondria in control and RNAi lines (exemplified by an ATG6 RNAi line). (E) Number of intact mitochondria per micrometer squared of the cytoplasm counted on the micrographs of meristematic, suspensor, and necrotic cells. Data represent the means ± SEM from three independent experiments, each including at least four cells per cell type. (F) Intact mitochondria (arrows) in a suspensor cell from the control line. (G) Swollen and degraded mitochondria (arrows) in a cell from an RNAi line. (H and I) Intracellular ATP content (H) and oxygen consumption (I). Data represent the means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01 (vs. control, Dunnett’s test). FW, fresh weight. (J and K) Cell vacuolization (J) and number of autophagosomes in the cytoplasm (K) estimated from the micrographs of meristematic (control line), suspensor (control line), and necrotic (ATG6 RNAi line) cells. Data represent the means ± SEM from three independent experiments, each including at least four cells per cell type. (L) Autophagosomes (open arrows) in the cytoplasm and autophagic bodies (closed arrows) in the vacuole of the embryo suspensor cell from a control line. (M and N) Quantification of necrotic death in control and RNAi lines after staining with FDA and FM4-64. (M) Staining patterns of EM and suspensor cells (control line) and necrotic cells (RNAi lines). DIC, differential interference contrast microscopy. (N) Frequency of necrotic cells. Data represent the means ± SEM from two independent experiments, each including two lines per genotype and ≥50 cells per line (P < 0.001; vs. control, Dunnett’s test). Different letters in D, E, J, and K indicate statistically different means (P < 0.001; Student’s t test). Bars: (A–C, main image) 5 µm; (C, inset) 0.5 µm; (F, G, and L) 2 µm; (M) 10 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal