Fig. 2: DON^5-FAM is transported via the EE and LE into lytic vacuoles.

a DON^5-FAM colocalized with RFP-AtALA1 in vesicles and EE-like structures in root hairs. In the vacuole of the root hair, DON^5-FAM colocalized with RFP-AtALA1 mainly in the tonoplasts. 35S::RFP-AtALA1 seedlings were treated with DON^5-FAM (9 μg/mL) for 12 h. Scale bar, 5 μm. b DON^5-FAM colocalized with RFP-RabA1d-labeled EE in root hairs. RFP-RabA1d, RFP-fused early endosome marker RabA1d. Six-day-old transgenic 35S::RFP-RabA1d seedlings were treated with DON^5-FAM (9 μg/mL) for 12 h. Scale bar, 5 μm. c DON^5-FAM was transported into γ-Tip-RFP labeled lytic vacuoles in the cells at the root meristem region. Six-day-old 35S::γ-Tip-RFP transgenic seedlings were treated with DON^5-FAM (9 μg/mL) for 12 h. Scale bar, 5 μm. d In root hair cells of proAtALA1::EGFP-AtALA1 transgenic Arabidopsis, EGFP-AtALA1 was localized to the PM and tonoplasts, as indicated by FM4-64 (8 μM) staining. Scale bar, 10 μm. e In root hair cells of transgenic Arabidopsis containing proAtALA1::EGFP-AtALA1 and 35S::RFP-RabA1d, EGFP-AtALA1 colocalized with the EE indicator RFP-RabA1d. Scale bar, 10 μm. f EGFP-AtALA1 colocalized with the LE indicator RFP-RabF2a in root hair cells of transgenic Arabidopsis containing proAtALA1::EGFP-AtALA1 and 35S::RFP-RabF2a. Scale bar, 10 μm. g Inhibition of TyrA23 (tyrphostin A23), BFA (brefeldin A), and Wm (wortmannin) on the transport of DON^5-FAM to vacuoles. Mock, wild-type seedlings were treated with DON^5-FAM (9 μg/mL) and FM4-64 (8 μM) for 12 h. TyrA23, BFA and Wm, Mock with TyrA23 (50 μM), BFA (50 μM) or Wm (33 μM) for 12 h. Scale bar, 5 μm. Experiments were independently repeated at least three times. The fluorescence density profile was 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).