Enhanced autophagy in the P. abies embryo suspensor. (A) Assessment of autophagic flux in the EM and suspensor cells using ConA treatment. Arrows denote autophagic bodies. Insets depict autophagosome docking to the vacuole (−ConA) and autophagic body (+ConA). N, nucleus; V, vacuole; asterisks, cell wall. Bars, 2 µm. (B) Typical autophagosome in the suspensor cell. Bar, 0.2 µm. (C) Number of autophagosomes in the cytoplasm estimated from the micrographs of EM and suspensor (S) cells. Data represent the means ± SEM from three independent experiments, each including at least four cells per cell type. (D) mRFP-Atg8 accumulates in puncta in the suspensor cells but remains cytoplasmic in the EM cells upon ConA treatment. Bars, 50 µm. (E) Correlation between anisotropic cell expansion (cell length), progression of cell death (mean FDA intensity; green symbols and green power trendline, R² = 0.5402), and accumulation of autophagosomes (number of mRFP-Atg8–positive puncta; red symbols and red polynomial trendline, R² = 0.8202) in the embryos. RFU, relative fluorescence units. (F) Accumulation of NBR1, mRFP-Atg8, and Atg8 upon ConA treatment detected by Western blot analysis of total protein extracts from the embryos. Actin was used as a reference control. Graph represents relative protein amounts shown as means ± SEM from three independent measurements. For each protein, the integrated band intensities were first normalized to corresponding intensities of Coomassie staining (loading) and then to the sample without ConA. The amounts of all three proteins were significantly increased upon ConA treatment (P < 0.001; Student’s t test).