Fig. 6

ANN5 interacts with RABE1b in plastidial nucleoids. Upper panel (a–b): Confocal optical section of N. benthamiana leaf epidermal plastid transiently expressing ANN5-YFP (a) and counterstained with DAPI (1 μg ml-1 for 15 min at room temperature) after fixation with 2% paraformaldehyde (24 h at 4 °C) (b). Pseudocolored fluorescence of (A) ANN5-YFP (yellow), (b) DAPI (magenta), (c) merged channels of ANN5-YFP and DAPI, and (d) overlaid with chlorophyll autofluorescence (blue). Lower panel (E–H): Confocal optical section of N. benthamiana leaf epidermal plastid transiently co-expressing ANN5-YFP (E) and RABE1b-CFP (f). Pseudocolored fluorescence of (e) ANN5-YFP (yellow), (f) RABE1b-CFP (magenta), (g) merged channels of ANN5-YFP and RABE1b-CFP, and (h) overlaid with chlorophyll autofluorescence (blue). See also Additional file 1: Figure S6. Scale bar = 10 μm. (j) FLIM-FRET analysis of interactions between ANN5 and RABE1b in plastidial nucleoids. Average CFP lifetime was measured in the donor leaf samples of N. benthamiana expressing only RABE1b-CFP and in the presence of acceptor in samples co-expressing RABE1b-CFP and ANN5-YFP. n = 7 individual epidermal cells. Measurements were performed on a single plastid per cell, ** indicates statistically significant differences (Student’s t-test, p < 0.05)