Fig. 5: GFP-ATG8 observation in At/At homo-grafting.
From: Autophagy is induced during plant grafting to promote wound healing
a–h Representative GFP fluorescence signals detected in grafting of At hypocotyls of GFP-ATG8 plants with a confocal laser scanning microscope. Maximum intensity projections of z-stacks. a,b Intact At. c,d 2 DAG. e,f 3 DAG. g,h 5 DAG. b, d, f, h Magnification images of dashed rectangle areas in (a, c, e, g). Yellow dashed lines indicate the grafting boundary. White arrowheads indicate autophagosome points. Bars = 50 µm. i–k Quantification of autophagosomes in the image of maximum intensity projections of (a, c, e, g). Error bars represent standard deviation. Asterisks indicate statistically significant differences compared with intact hypocotyls as determined by one-way ANOVA (P < 0.05) followed by Dunnett’s multiple comparison test (* P < 0.05, ** P < 0.01; n = 12). l Fluorescent image of GFP-ATG8 observed in a ring shape, a characteristic structure of autophagosome. Bars =100 µm. A magnified image of the bright spot pointed by the arrowhead is shown in inset. Bar =20 µm. m–p Accumulation of GFP vesicles in the vacuoles upon concanamycin A treatment. m, n Images of the scion side of the grafted hypocotyl. o, p Images of the rootstock side of the grafted hypocotyl. Dashed rectangles in (m, o) indicate the areas of (n, p), respectively. White arrowheads indicate autophagic body in the vacuoles. Bars =100 µm (m, o) and 50 µm (n, p). q Representative data of ATG8 cleavage in At hypocotyl grafting using an anti-GFP antibody. The top panel shows the signal corresponding to GFP-ATG8 and the middle panel shows the ATG8-cleavaged GFP signal. The bottom panel shows a membrane-stained image with Ponceau S to ensure uniformity of loading. The numbers at the bottom of the middle panel show the relative intensity of ATG8-cleavaged GFP at each time when the signal intensity of intact is set to 1.00. All experiments were performed three times independently.
