Figure 6.

HAP13 is essential for BFA-sensitive post-Golgi trafficking. A to D, Epidermal cells of 4-DAG wild-type roots were pulse labeled with 4 µm FM4-64 and incubated in MS medium for 1 min (A), 15 min (B), 90 min (C), or 3 h (D) before visualization. E and F, Epidermal cells of 4-DAG wild-type roots were pulse labeled with 4 µm FM4-64 and incubated in MS medium supplemented with BFA for 50 min before imaging (E). BFA washout followed by 1 h of incubation in MS medium is shown in F. G to J, Epidermal cells of 4-DAG hap13-1 roots were pulse labeled with 4 µm FM4-64 and incubated in MS medium for 1 min (G), 15 min (H), 90 min (I), and 3 h (J) before visualization. K and L, Epidermal cells of 4-DAG hap13-1 roots were pulse-labeled with 4 µm FM4-64 and incubated in MS medium supplemented with BFA for 50 min before imaging (K). BFA washout followed by 1 h of incubation in MS medium is shown in L. M to R, TEM of wild-type (M, O, and Q) and hap13-1 (N, P, and R) root epidermal cells under control conditions (M and N), treated with 50 µm BFA for 50 min (O and P), or treated with BFA followed by a 1-h washout (Q and R). Arrows in O and P indicate the Golgi apparatus surrounding the BFA compartment. S to U, Quantification of FM4-64 uptake (S), percentage of fluorescence distribution (T), and number of BFA compartments (U) either upon BFA treatment (50 µm for 50 min) or after BFA washout (1 h). Data were collected from nine optical sections of roots in which six to eight cells in each optical section were used to calculate the areas of fluorescence or the number of punctate vesicles with ImageJ. Results shown in S to U are given as means ± se. Asterisks represent significant differences (Student’s t test, *P < 0.01 or ***P < 0.001). Nonbiased double-blind analyses were conducted to confirm the differences between the wild type (WT) and hap13-1. Bars = 7.5 µm for A to L, 200 nm for M, N, Q, and R, and 500 nm for O and P. [See online article for color version of this figure.]