Figure 1.
Characterization of vamp721vamp722 mutants.
(A) Schematic structures of VAMP721 and VAMP722 genes. Orange boxes represent exons and blue boxes represent UTRs. The sites of T-DNA insertions are indicated. (B) RT-PCR from RNA extracts of the vamp721vamp722 mutants. The expression of VAMP721 and VAMP722 was not detected in the double mutants. Two biological replicates were performed. (C) 5-d-old wild-type and vamp721vamp722 mutant seedlings derived from vamp721+/-vamp722-/- plants are shown. Bars = 2 mm. (D) and (E) Root tip regions in 5-d-old wild-type (D) and vamp721vamp722 mutant seedlings (E) stained with propidium iodide. Note the disorganized cell files in the mutant. Bars = 20 µm. (F) 5-d-old wild-type and vamp721vamp722 mutant seedlings under dark growth are shown. Bars = 500 µm.
Figure 2.
Cell wall stubs and incomplete cytokinesis in vamp721vamp722 mutants.
(A) and (B) Toluidine blue O-stained root longitudinal sections of wild-type (A) and vamp721vamp722 seedlings (B). Cell wall stub (arrowhead) and gap (arrow) were observed in double mutants. Bars = 20 µm. (C) and (D) Confocal images of propidium iodide-stained nuclei (red) and Calcofluor-stained cell walls (green) in root tip cells of 3-d-old seedlings. vamp721vamp722 mutants showed incomplete cytokinesis (D) compared with that of wild type (C). Arrowheads indicate two nuclei in a single cell; arrow indicates discontinuous cell wall. Bars = 10 µm. (E–G) Epidermal cells of cotyledons visualized using propidium iodide staining. Compared with wild type (E), cell wall stubs were observed in cotyledons of mutants (F), which was confirmed by 3D projection (G). Bars = 20 µm. (H) and (I) Time-course analysis of growing cell plates stained with FM4-64 in root tips of 3- to 5-d-old wild-type (H) and vamp721vamp722 (I) seedlings. Arrows indicate the growing or arrested cell plates. Bars = 5 µm (H); 10 µm (I).
Figure 3.
GFP-tagged VAMP721 and VAMP722 are localized to the cell plates and postcytokinetic walls.
(A) and (C) GFP-VAMP721 (A) and GFP-VAMP722 (C) label the expanding cell plates (arrowheads) and postcytokinetic walls (arrows), which are merged with the membrane labeled with FM4-64. Bars = 10 µm. (B) and (D) Time series of growing cell plates in root tip cells stained with FM4-64. GFP-VAMP721 (B) and GFP-VAMP722 (D) signals are localized to the cell plates during the progression of cytokinesis. Bars = 5 µm.
Figure 4.
Cell plate formation during cytokinesis in control, vamp721vamp722 and complemented double mutant root tips.
Confocal analysis of cell plate formation monitored by GFP signals (green) together with FM4-64 staining (red) in cytokinetic root tip cells. White arrows indicate abnormal cell plates with irregular direction and/or thickness; yellow arrows indicate asymmetric expansion of cell plate and pink arrows indicate symmetric expansion or complete cell plate formation. (A–C) The cell plate formation in GFP-KNOLLE seedlings used as the control. Bars = 10 µm. (D–F) The cell plate formation in vamp721vamp722 seedlings labeled with cell plate marker GFP-KNOLLE. Bars = 10 µm. (H–J) The cell plate formation in complemented double mutant seedlings rescued by pVAMP721::GFP-VAMP721. Bars = 10 µm.
Figure 5.
VAMP721- and VAMP722-labled organelles are distinct from the Golgi and PVC.
(A–D) 5-d-old seedlings were pre-incubated in 50 µM BFA for 30 min before incubation in 50 µM BFA plus 5 µM FM4-64 for another 30 min. (A) and (B) GFP-VAMP721 (A) and GFP-VAMP722 (B) relocated to BFA compartments labeled with FM4-64. Bars = 10 µm. (C) VHA-a1-GFP-labeled TGN was sensitive to BFA treatment and colocalized with BFA compartment from FM4-64. Bars = 10 µm. (D) FM4-64 was accumulated into the core of BFA compartment surrounded by the Golgi marker N-ST-YFP after BFA treatment. Bars = 10 µm. (E–G) Seedlings were incubated with 33 µM wortmannin for 60 min. DMSO was used as the control. Bars = 10 µm. (E) In contrast to the control, the PVC marker GFP-RabF2b was induced to form small vacuoles after wortmannin treatment. Bars = 10 µm. (F) and (G) Similar to the DMSO control, the organelles labeled with (F) GFP-VAMP721 and (G) GFP-VAMP722 were not changed following wortmannin treatment. Bars = 10 µm. (H–K) Confocal laser scanning microscopy (CLSM) analysis of seedling root epidermal cells co-expressing the Golgi marker N-ST-YFP or PVC marker GFP-RabF2b as indicated (green) and mCherry-VAMP721 or mCherry-VAMP722 as indicated (red). Co-localization analysis showed that mCherry-VAMP721- and mCherry-VAMP722-labeled organelles were closely associated with Golgi stacks in (H) and (J). Similarly, as shown in (I) and (K), mCherry-VAMP721- and mCherry-VAMP722-labeled organelles were also distinct from the PVC marker GFP-RabF2b. Bars = 5 µm.
Figure 6.
VAMP721- and VAMP722-labeled organelles are rapidly labeled by internalized FM4-64 and overlap the VHA-a1 TGN.
(A–E) Root tip cells expressing VHA-a1-GFP (A), GFP-RabF2b (B) and (C), GFP-VAMP721 (D), or GFP-VAMP722 (E) (each green) incubated with FM4-64 (red) for the times indicated. Pink arrows indicate GFP-labeled regions without detectable FM4-64 labeling; yellow arrows indicate FM4-64–labeled regions without detectable GFP signal; white arrows indicate regions with extensive colocalization between GFP and FM4-64 signals. Bars = 5 µm. (F) and (G) Confocal laser scanning microscopy analysis of root epidermal cells coexpressing VHA-a1-GFP (green) and (F) mCherry-VAMP721 (red) or (G) mCherry-VAMP722 (red). Bars = 5 µm.
Figure 7.
Concanamycin A interferes with cell plate formation.
(A–F) Fluorescence imaging of root cells. The cell outlines were stained with FM4-64. Compared with the expanding cell plates in controls (A), (C) and (E), ConcA treatment for 2 h induced intracellular accumulation of GFP-KNOLLE (B), GFP-VAMP721 (D), and GFP-VAMP722 (F), and prevented the cell plate formation indicated with arrowheads in (B), (D), and (F). Bars = 5 µm.
Figure 8.
Localization of the plasma membrane and tonoplast markers in control and vamp721vamp722 mutant seedlings.
(A) GFP-Lti6a localization in control root. Note (enlargement at right) that GFP-Lti6a remained extensively colocalized with FM4-64 at the plasma membrane. Bars = 10 µm. (B) GFP-Lti6a localization in vamp721vamp722 roots. Note (enlargement at right) that GFP-Lti6a abnormally accumulated in the cytoplasm. Bars = 10 µm. (C) PIP2A-GFP localization in control roots. Note (enlargement at right) that PIP2A-GFP remained extensively colocalized with FM4-64 at the plasma membrane. Bars = 10 µm. (D) PIP2A-GFP localization in vamp721vamp722 roots. Note (enlargement at right) that PIP2A-GFP accumulated inside aberrant intracellular compartments. Bars = 10 µm. (E) and (F) TIP1;1-GFP showed similar tonoplast labeling patterns in cells of control (E) and vamp721vamp722 (F) root tips. Bars = 10 µm. (G) and (H) TIP1;1-GFP-labeled tonoplast and small vacuoles in cells of control (G) and vamp721vamp722 (H) hypocotyls. Red signals represent chloroplast autofluorescence. Bars = 10 µm.
Figure 9.
Hypothetical model of vesicle trafficking during cell plate formation in root tip cells of Arabidopsis.
Based on this study and previous results, secretory (black arrow) and endocytic (red arrow, such as FM4-64) trafficking pathways converge at the TGN/EE to reach the cell plate. VAMP721 and VAMP722 compartments mediate the traffic of Golgi-derived vesicles to cell plate via TGN/EE membrane domain. In addition, VAMP721 and VAMP722 compartments mediate protein secretion to the plasma membrane. PM, plasma membrane; TGN, trans-Golgi network; EE, early endosome; PVC, prevacuolar compartment.