Figure 4.

Impaired Trafficking of PIN2-GFP in clc2-1 clc3-1.

(A) to (D) BFA-induced internalization of PIN2-GFP in the wild type and clc2-1 clc3-1.

(A) CHX treatments for 30 min.

(B) and (C) CHX pretreatments for 30 min followed by washout with CHX and BFA for 15 min (B) and 60 min (C).

(D) The average number of PIN2-GFP–labeled BFA bodies ([B] and [C]).

(E) to (H) Visualization of vacuolar accumulation of PIN2-GFP in dark-treated seedlings of the wild type and clc2-1 clc3-1.

(E) Dark treatments for 0 h.

(F) Dark treatments for 2 h.

(G) Dark treatments for 4 h.

(H) The average relative intensity of PIN2-GFP in vacuoles for each cell ([F] and [G]).

(I) to (U) Endocytic recycling of PIN2-GFP in the wild type and clc2-1 clc3-1. The seedlings were pretreated with CHX for 30 min, followed by washout with CHX and BFA for 60 min, and finally by washout with 0.5× MS liquid media for different lengths of time (0, 10, 20, 30, 40, and 60 min) in the wild type ([I] to [N]) and clc2-1 clc3-1 ([O] to [T]) before CLSM imaging.

(U) The average number of PIN2-GFP–labeled BFA bodies after BFA removal in the wild type and clc2-1 clc3-1.

Arrowheads show PIN2-GFP–labeled BFA bodies or vacuoles. Bars = 10 µm. Shown are means ± sd. Triple asterisks indicate P < 0.001 (D) or 0.0001 (H), respectively (Student’s t test).

[See online article for color version of this figure.]