Deficiencies of PLDs result in promoted autophagy. (A and B) MDC staining (left) and quantification of puncta per root section (right) of WT, pldα1, pldζ1, pldβ1, pldδ (A) and PLDδ-OE (B). Five-day-old seedlings were treated with Con A (0.5 μM), followed by staining with MDC. The labeled autophagosomes of root cells were visualized by fluorescence confocal microscopy using a DAPI-specific filter and representative images were shown (bars: 50 μm). Numbers of puncta per root section were measured and data were shown as mean ± SD (n = 20). Statistical significance was determined by Student's t-test (***, P < 0.001, compared to WT). (C and D) TEM analyses (left, bars = 50 μm) and quantification of autophagic structures (right) in WT and pldα1, pldζ1, pldβ1, pldδ seedlings (C), and seedlings overexpressing PLDδ (D). Three-week-old seedlings were treated with Con A (0.5 μM) and mesophyll cells were observed. “A” indicates the autophagic structures. CW, cell wall; C, chloroplast; M, mitochondrion; V, vacuole. Experiments were biologically repeated for three times and data were shown as mean ± SD (n = 10). Statistical significance was determined by Student's t-test (***, P < 0.001, compared to WT).