PMC

(A) Alignment of VPS45 protein sequences from various organisms. At: Arabidopsis thaliana, (At1g77140); Pp: Physcomitrella patens (XP_001758633); Dd: Dictyostelium discoideum (XP_635835); Sc: Saccharomyces cerevisiae (NP_011420); Hs: Homo sapiens (NP_009190). (B) Complementation by VPS45-GFP of ben2 mutant phenotypes. After BFA treatment (25 µM for 2 h), PIN1 accumulated in intracellular compartments in wild type root vascular tissue. Whereas intracellular accumulation of PIN1 was less pronounced in ben2 mutant, clear accumulation of PIN1 was detected in ben2 mutant harboring VPS45-GFP. Similarly, PIN2 accumulation in root epidermal cells upon BFA treatment was recovered in ben2; VPS45-GFP. Scale bar: 10 µm.

(A,B) Antibody staining of endogenous PIN1 protein in vascular tissue (A) and PIN2 on epidermal cells (B) of wild type and ben1; ben2 double mutant roots. Arrows indicate polar localization of PIN proteins. (C,D) ben mutations and chemical inhibitor of auxin transport collectively diminish auxin response maxima. Auxin response maxima were visualized by DR5rev::GFP with color coding of signal intensities. Seedlings of indicated genotypes (3d) were transferred to mock (C) or NPA-containing media (50 µM) and grown for 2 days before imaging. Scale bars: 20 µm for (A,B); 50 µm in (D) for (C,D).

(A) Wild type VPS45-GFP (green) colocalized with FM4-64 (red) within 10 minutes after the onset of staining. Additional endocytic compartments were labeled by prolonged incubation (upper panels). Mutated VPS45-GFP carrying ben2 mutant sequence (VPS45D129N-GFP, green) did not colocalize with FM4-64 under the same conditions (lower panels). (B) Immunostaining of BEN1 (red) and VPS45-GFP (green). Whereas wild type version of VPS45-GFP partially colocalized with BEN1 (upper left panel), ben2 mutation abolished the colocalization (lower left panel). BEN1 and VPS45-GFP responded differently to BFA (upper right panel). Whereas BEN1 accumulates to the center of the BFA compartment in BFA-treated cells, majority of VPS45-GFP localized to the periphery of the BFA compartment. ben2 mutation caused mislocalization of the VPS45-GFP protein (green), although it did not affect the agglomeration of BEN1 signal (lower right panel). Magnified views of the regions indicated by white squares are shown in the bottom panels. The right panels show merged images. Scale bars: 5 µm.

(A,B) PIN2 immunofluorescence signals in root epidermal cells without drug treatment (A) and after ES1 treatment (36 µM, 2 h) (B). Arrowheads indicate intracellular agglomerations of PIN2 signals. (C) Quantitative analyses of intracellular accumulation of PIN2 in ES1 treated root epidermal cells. Asterisks indicate significant difference from wild type control (**: P<0.01; ***: P<0.0001 by t-test). Error bars indicate standard deviation among individual roots. N: number of cells examined. (D) Uptake of endocytic tracer FM4-64 in wild type (left) and ben2 (right) root epidermal cells, 15 minutes after the onset of FM4-64 labeling. Magnified views of the boxed regions are indicated. Signal intensity is represented by the color code as indicated. (E) Ultrastructure of membranes associated with the Golgi apparatus in root epidermal cells of wild type and mutants. Scale bars: 20 µm in (B) for (A,B); 5 µm for (D); 300 nm for (E).

(A,B) Localization of PIN1-GFP in developing LRPs. PIN1-GFP localizes to the anticlinal sides of young LRPs (white arrowheads) and gradually shifts its localization toward the tip of developing LRP within three days (A; yellow arrows). Relocation was less clear in the ben1; ben2 double mutant (B). (C,D) Auxin response maxima as visualized by DR5rev::GFP reporter. Whereas sharp peaks were formed at the tips of LRPs in wild type, DR5 expression was broader in malformed LRPs of ben1; ben2 three days after the onset of induction (C). Sharp peaks of auxin response maxima were maintained over time in wild type. In contrast, new auxin response maxima were generated in the base of LRPs in ben1; ben2 (D). (E) A model to explain altered root architecture. PIN1 relocates towards the tip of LRP and facilitates directional auxin flow in provascular tissue (green arrows), resulting in generation of auxin maxima at the tip of LRP (green circle). PIN relocation is compromised in ben1; ben2 double mutant. PIN1 is ectopically expressed in the epidermis by unknown mechanism, which in turn generates atypical auxin flow (blue arrows). Scale bars: 20 µm in (B) for (A,B); 50 µm in (C); 100 µm in (D).