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Science 337 (6100): 1333-1336

Copyright © 2012 by the American Association for the Advancement of Science

Initiation of Cell Wall Pattern by a Rho- and Microtubule-Driven Symmetry Breaking

Yoshihisa Oda1,2,*, and Hiroo Fukuda1,*

1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
2 Japan Science and Technology Corporation (JST), PRESTO, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan.


Figure 1
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Fig. 1. Active ROP11 is localized in secondary wall pits to anchor MIDD1. (A) tagRFP-MIDD1 and GFP-ROP11 in differentiating xylem cells. Both signals are substantially overlapped (white arrowheads). (B) Secondary wall pattern (WGA) of a cell expressing GFP-ROP11G17V or GFP-ROP11Q66L. (C) Percentages of cells that developed flat secondary walls without pits. Data are means ± SD of three experiments (n > 200 cells), *P < 0.01 [analysis of variance (ANOVA) with Scheffe test]. (D) Confocal cross-sections of cell cortex in a differentiating xylem cell expressing nYFP-ROP11 and cYFP-MIDD1{Delta}N. The intensity plots of BiFC and WGA along the plasma membrane between red arrowheads are shown below the images. Scale bars indicate 5 μm; a.u., arbitrary units.

 

Figure 2
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Fig. 2. ROPGAP3 and ROPGEF4 localize preferentially in secondary wall pits in differentiating xylem cells. (A and B) Accumulation of GFP-GAP3 [(A), yellow arrowheads] and GFP-ROPGEF4PRONE [(B), red arrowheads] at the plasma membrane in the secondary wall pits. (C) Accumulation of GFP-ROPGEF4PRONE at the plasma membrane (cyan arrowheads) in early differentiating xylem cells. (D) Accumulation of GFP-ROPGEF4PRONE (green) at the center of pits (white arrowheads) of the secondary wall (magenta). (E) Double labeling of GFP-ROPGAP3 (green) and tagRFP-ROPGEF4PRONE (magenta) in differentiating xylem cells (top). Intensity profile between the orange arrowheads (bottom). (F) Distance between the edge of GFP signals and WGA signals. Values are means ± SD (n = 7 pits), *P < 0.01 (t test). The location of GFP-ROPGEF4PRONE is more central than that of GFP-ROPGAP3. Scale bars, 5 μm.

 

Figure 3
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Fig. 3. Reconstitution of the active ROP domains and local microtubule disruption in nonxylem cells. (A to C) Localization of ROPGEF4 PRONE and microtubules in nonxylem Arabidopsis cultured cells. Areas encircled by yellow dotted lines are magnified below. ROP11 and ROPGAP3 were coexpressed with tagRFP-MIDD1 and GFP-ROPGEF4PRONE (A); tagRFP-MIDD1, tagRFP-ROPGEF4PRONE, and GFP-TUB6 (B); or tagRFP-ROPGEF4PRONE and GFP-TUB6 but not tagRFP-MIDD1 (C). Without MIDD1, cortical microtubules are not depolymerized around tagRFP-ROPGEF4 PRONE clusters (C). Scale bars, 5 μm.

 

Figure 4
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Fig. 4. Cortical microtubules restrict the localization of the active ROP11-MIDD1 complex. (A to D) Shape of tagRFP-MIDD1–labeled plasma membrane domains reconstituted in leaf epidermal cells of N. benthamiana coexpressing ROPGEF4PRONE, ROP11, and ROPGAP3. Yellow dotted circles indicate plasma membrane domains with tagRFP-MIDD1. (A) Treated with mock. (B) Treated with 30 μM oryzalin. (C) Coexpressed with AtKinesin-13A. (D) Coexpressed with AtKinesin-13A{Delta}M (inactive form). (E) Circularity (2{pi} x area/perimeter2) of the plasma membrane domains marked with tagRFP-MIDD1 in leaf epidermal cells. Values are means ± SD (n > 300 domains), *P < 0.01 (ANOVA with Scheffe test). (F) Visualization of cortical microtubules (GFP-TUB6) and plasma membrane domains (tagRFP-MIDD1) in leaf epidermal cells coexpressing ROP11, ROPGAP3, and ROPGEF4PRONE. Cortical microtubules run along the borders of the plasma membrane domains (arrowheads). Scale bars, 5 μm.

 


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