Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Expression of Arabidopsis ARF1 and Its Homologs (A-E) Expression of the GUS and ERYFP reporter genes under the control of the protmoters of the indicated ARF homologs. Flower (A). Primary roots (B-E). (F) Expression of the ERYFP reporter gene under the control of the ARF1 promoter. (G-K) Expression of the GUS reporter gene under the control of the ARF1 promoter. 3-day-old seedling (G). Cotyledon (H); inset: stomata. Leaf primordium and trichome cells (I). Flower (J). Embryos at different developmental stages (K1-7; 32-cell, globular, early, mid, later heart-shaped, torpedo and mature embryo). (L-P) Expression of arf1-ARF1-WT-EGFP in Arabidopsis transgenic lines. In primary root cells, overview (M). GFP fluorescence may be at the position of the forming cell plate (M; arrow). Localizes to growing root hairs (N; arrows). In stomata (O) and trichome cells (P). PI-staining (Red) was used to label root cell structures in (L,M). Bars = 200μm in (A), (H) to (J), 50μm in (B) for (B) to (E) and (L), 25μm in (K1) to (K7), 10μm in (M) to (O) and 50μm in (P).
• Supplemental Figure 2 - Co-localization Analysis of Golgi and Endocytic Markers in onion and Arabidopsis cells (A1-3) Co-localization analysis of NAG-ECFP (A1; green) and N-ST-YFP (A2; red) in onion epidermal cells. Note that all NAG-ECFP fluorescence coincides with N-ST-YFP fluorescence. (B1-3) Co-localization analysis of NAG-EGFP (B1; green) and endocytic marker FM4-64 (B2; red) in onion epidermal cells. No co-localization was observed between NAG-EGFP and FM4-64. (C1-3) Co-localization analysis of 35S-ARF1-WT-ECFP (C1; green) and ARA6-EYFP (C2; red) in onion epidermal cells. 35S-ARF1-WT-ECFP displays no co-localization with ARA6-EYFP. (D1-3) Co-localization analysis of NAG-EGFP (D1; green) and endocytic marker FM4-64 (D2; red) in meristematic root cells. Two-photon microscopy showed that NAG-GFP fluorescence is often associated with but most likely not overlapping with FM4-64 signal. (E1-3) Co-localization analysis of NAG-EGFP (E1; green) and FM4-64 (E2; red) at 20min of incubation with 100 μM BFA. Note BFA caused aggregations of FM4-64 with some of the NAG-EGFP signals which are surrounded by aggregates only labeled by NAG-EGFP. Bars = 5μm in (A1) for (A1) to (D3), 10μm in (E1) for (E1) to (E3).
• Supplemental Figure 3 - ARF1-Q71L and ARF1-T31N Expression in the Arabidopsis Root Meristem Interferes with Root Growth, Cell Expansion and Cytokinesis (A) Expression of the GUS reporter gene under the control of the RCH1 promoter. (B,G,K) Arabidopsis primary root with ARF1-WT overexpression in root meristem. Inset in (K) shows normal ERGFP expression in meristmatic root cell. (C-D,H,L-N) Arabidopsis primary root with ARF1-Q71L expression in root meristem. Arrows in (C,H) point to disorganized root meristems. Arrows in (D) point to the differentiated vascular bundles and ectopic starch granules. Inset in (D) indicates columella mis-specification (arrow) visualized by histochemical analysis of a columella-specific GUS marker. Cell expansion and cytokinesis defects in boxed area of (L) were affected. ERGFP expression in (M) was altered and multinucleate cells in root meristem are marked by H2B-YFP expression (N). (E-F,I-J,O-Q) Arabidopsis primary root with ARF1-T31N expression in root meristem. ERGFP fluorescence was altered in (O,P). Multinucleate cells in root meristem are marked by H2B-YFP expression (Q). PI-staining (Red) was used to label root cells in (B,C,H,K-Q). Green fluorescence in the images is from rch1GV induced ERGFP expression in root meristem. Bars = 50μm in (A), 200μm in (B) for (B), (C), (E), (H) and (I), 50μm in (G) for (D), (F), (G) and (J), 50μm in (K) for (K), (L) and (O), 10μm in (M) for (M), (N), (P) and (Q).
• Supplemental Movie 1 - Co-localization analysis of 35S-ARF1-WT-ECFP (green) and 35S-ARF1-Q71L-YFP (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels
• Supplemental Movie 2 - Co-localization analysis of NAG-ECFP (green) and N-ST-YFP (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 3 - Co-localization analysis of 35S-ARF1-WT-ECFP (green) and N-ST-YFP (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 4 - Co-localization analysis of NAG-ECFP (green) and 35S-ARF1-WT-EYFP (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 5 - The fluorescence of 35S-ARF1-WT-EGFP (green) and endocytic marker FM4-64 (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 6 - Co-localization analysis of NAG-EGFP (green) and endocytic marker FM4-64 (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 7 - Co-localization analysis of 35S-ARF1-WT-EGFP (green) and RFP-ARA7 (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 8 - Co-localization analysis of 35S-ARF1-WT-ECFP (green) and ARA6-EYFP (red) in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.
• Supplemental Movie 9 - The fluorescence of 35S-ARF1-WT-EYFP is associated with the cortical network of actin filaments marked by EGFP-TALIN in onion epidermal cells. Time-lapse movies were taken at 1.686 second intervals and recorded at a rate of 5 full frames (uncompressed) per second with a resolution of 400×400 pixels.