Fig. 3. CIA^FITC is transported via the EE and LE into lytic vacuoles.

a CIA^FITC colocalized with AtALA7-RFP in vesicles and vacuoles of the root hair. 35S::AtALA7-RFP seedlings were treated with CIA^FITC (15 μg/mL) for 6 h. Scale bar, 5 μm. b CIA^FITC and FM4-64 fluorescence in root cells of wild-type Arabidopsis at 180 min, 183 min, and 201 min after treatment with CIA^FITC (15 μg/mL) and FM4-64 (8 μM). Scale bar, 10 μm. The triangle indicates the location of CIA^FITC aggregates. c Distribution of CIA^FITC in root cells of transgenic Arabidopsis 35S::γ-Tip-RFP. Seedlings were treated with CIA^FITC (5 μg/mL) for 6 h; CIA^FITC fluorescence mainly accumulated in lytic vacuoles. Scale bar, 10 μm. d Inhibition of TyrA23, BFA, and Wm on the accumulation of CIA^FITC in vacuoles of root cells. Arabidopsis wild-type seedlings were treated with 5 μg/mL CIA^FITC and 8 μM FM4-64 (Mock), and extra TyrA23 (50 μM), BFA (50 μM) or Wm (33 μM) for 6 h. Scale bar, 10 μm. e AtALA7-eGFP was localized to the PM and tonoplasts in root hair cells of proAtALA7::AtALA7-eGFP transgenic Arabidopsis, which were stained with FM4-64 (8 μM). proAtALA7, the native promoter of AtALA7. Scale bar, 10 μm. f AtALA7-eGFP colocalized with the EE indicator RFP-RabA1d in root hair cells of transgenic Arabidopsis containing proAtALA7::AtALA7-eGFP and 35S::RFP-RabA1d. Scale bar, 10 μm. g AtALA7-eGFP colocalized with the LE marker RFP-RabF2a in root hair cells of transgenic Arabidopsis containing proAtALA7::AtALA7-eGFP and 35S::RFP-RabF2a. Scale bar, 10 μm. Experiments were independently repeated at least three times. The fluorescence density profiles were measured along the dotted lines using ImageJ. The values of the Pearson correlation coefficient (Rp) and the Spearman correlation coefficient (Rs) show the extent of colocalization between the two target molecules. The values range between +1 (positive correlation) and −1 (negative correlation).