Figure 8.

ES7 specifically affects callose deposition at the cell plate and no other cellular components. The effect of ES7 and other known cytokinesis inhibitors on the localization of subcellular markers and callose deposition is compared. A to E, YFP-RABA2A-labeled cells after drug treatments. Under DMSO treatment, YFP-RABA2A accumulates at the leading edge of the expanding cell plate at a 100% frequency (A). Under ES7 treatment, YFP-RABA2A and FM4-64 accumulation shows characteristic cell plate gaps at an 87% frequency (B; white arrow). Under caffeine treatment, the accumulation of YFP-RABA2A is highly irregular at the cell plate in 85% of scorable cells (C). Flufenacet treatment causes cell plate gaps, similar to those under ES7 treatment in 57% of scorable cells. In addition, aggregates of RABA2A are observed under the same treatment (D; yellow arrow). ConcA causes cell plate mislocalization and fluorescent aggregates in 88% of scorable cells (E). F to J, Dividing GFP-MAP4-labeled cells after chemical treatment. Under DMSO, the regular phragmoplast structure of GFP-MAP4 labeling is observed at a 100% frequency (F). Under ES7 treatment, the phragmoplast arrangement remains unaffected in 100% of scorable cells (G). Caffeine treatment causes abnormal phragmoplast structure in 88% of scorable cells (H). Under flufenacet treatment, GFP-MAP4 labels ectopic phragmoplast-like structures at the center of the mature cell plate in 60% of scorable cells (I). Under ConcA treatment, regular phragmoplast GFP-MAP4 labeling is observed at a 100% frequency (J). K to O, GFP-CESA3-labeled cells under chemical treatment. Under DMSO treatment, GFP-CESA3 weakly labels the mature cell plate, with high accumulation of vesicles localizing at the cell plate vicinity in 100% of scorable cells (K). ES7 treatment reduces GFP-CESA3 accumulation at the cell plate in 100% of scorable cells, and diffused fluorescent localization indicating vacuolar localization is observed (L; yellow arrow). Under caffeine treatment, GFP-CESA3 shows a strong aggregate pattern with partial localization of the aggregates at the cell plate stub in 99% of scorable cells (M; white arrow). Flufenacet treatment causes severe aggregation of GFP-CESA3 vesicles in 100% of scorable cells (N). ConcA treatment causes strong vacuolar localization of GFP-CESA3 in 100% of scorable cells (O). P to S, Immunostaining of callose after chemical treatment. Under DMSO treatment, cell plate accumulation of callose is observed at a 100% frequency (P). Under ES7 treatment, callose deposition is absent at the cell plate at a 100% frequency (Q). Similarly, caffeine treatment also reduces callose deposition in 75% of scorable cells (R). Flufenacet treatment does not affect callose deposition at the cell plate in all observed cells (S). Concentrations of chemicals are given in brackets (in μm). The respective marker fluorescence is shown in green, while FM4-64 staining of plasma membrane (A–D) and PI staining of the cell wall (F–O) are shown in magenta. Callose labeling is shown in red, while nuclei staining by DAPI is indicated in blue. Results were observed from more than 36 cells from at least four roots of each marker plant for each drug treatment. Bars = 10 µm.