Fig 1.
Jigsaw-puzzle pattern formation by leaf epidermal cells in Arabidopsis thaliana.
(a) Traced image of epidermal cell wall in an abaxial cotyledon surface. Cell contours were visualized with the plasma membrane marker GFP-PIP2a on the cotyledon surface of seedlings 2 days (left), 4 days (middle) and 7 days (right) after sowing. Scale bar = 100 μm. (b) Time sequential images of abaxial cotyledon epidermal cells. Scale bar = 10 μm.
Fig 2.
(a) Governing equation of the model. (b) Initial distribution of cell wall taken from measurements of seedlings. Black indicates the cell wall; white indicates the cell interiors. Red asterisk indicates a small stomatal lineage cell. (c) Cell wall interdigitation at t = 100. Red asterisk indicates a small stomatal lineage cell. (d) Signaling molecule concentration at the interface. The protruded side had a higher ROP2/ROP6 ratio. Cyan indicates ROP2 activity; magenta indicates ROP6 activity. (e) Dispersion relation of the model obtained by a linear stability analysis. The specific wavenumber component grew. (f) The ratio of wavenumber components obtained by analysis of in vivo cell wall pattern changes. The upper leaf regions of 2-, 4-, and 7-day-old seedlings were analyzed. The red line indicates the ratio between 4- and 2-day-old seedlings in each wavenumber. The green line indicates the ratio between 7- and 2-day-old seedlings in each wavenumber.
Fig 3.
Effects of decreased cellulose on cell wall thickness.
(a) Transmission electron microscopic images of the cotyledon lateral cell wall in an rsw2/kor1 mutant, in which cellulose production is decreased. The red line shows the thickness of the cell wall. Scale bars = 200 nm. (b) The mean thickness of the cotyledon lateral cell wall in the wild type (Col-0) and rsw2/kor1 mutant plants 7 days after sowing. Data are mean ± SD (n = 5). **p < 0.005 (U-test). Note that thickness increased in the rsw2/kor1 mutant. (c) Transmission electron microscope images of the cotyledon lateral cell wall in wild-type seedlings with or without cellulose treatment. The red line shows the thickness of the cell wall. Scale bars = 200 nm. (d) The mean thickness of the cotyledon lateral cell wall after 7 days in wild type plants (Col-0) with or without cellulase treatment. Data are mean ± SD (n = 6). *p < 0.01 (U-test). Note that thickness was increased by cellulase treatment. (e) A numerical simulation of the model in which the effective range of signaling molecule was changed. The thickness of the cell wall increased by increasing the effective range of action of signaling molecule. (f) Model of cell wall thickness change. A certain point of the cell wall-cytoplasm interface detects the amount of cytoplasm around that point, and its detection range is defined by the kernel diameter (blue circle). The boundary reaches a steady state when the interface speed becomes zero due to the signaling molecule effect from the neighboring cell. If the kernel diameter is larger (red circle), the effect comes from further away and, the steady-state thickness increases as a result.
Fig 4.
Effects of cellulose degradation on cell wall interdigitation.
(a) The rsw2/kor1 mutant displayed less curvature. Scale bars = 50 μm. (b) Effects of cellulase treatment on curvature formation. Curvature formation was also inhibited. Scale bars = 50 μm. (c) A numerical simulation of the model with a different range of action of signaling molecule. Less interdigitation occurred when the signaling molecule range of action increased, which is consistent with experimental observations.
Fig 5.
Characteristics of cell wall three-way junctions.
(a) Time-lapse observation of a three-way junction using the membrane marker GFP-PIP2a. The angles were mostly 120°. Scale bar = 10 μm. (b) The relationship between cell size and the degrees of three-way junctions 7 days after sowing. Larger, more developed cells were more likely to have angles closer to 120°. RMSD, root-mean-square deviation. See also S3 Fig for data from samples 2 and 4 days after sowing. (c) Snapshots of the numerical simulation of the model at a three-way junction. The numerical simulation shows that the angles of a three-way junction gradually approach 120°.
Fig 6.
A three-way junction became a forced cytoplasmic protrusion and may affect downstream molecular pathways.
(a) Maximum intensity projection of GFP-tubulin in cotyledon leaf epidermal cells. White arrows and red arrowheads indicate pavement interdigitation and a three-way junction, respectively. Scale bar = 10 μm. (b, c) A representative circle with a radius of 5 μm centered at an interdigitation point (b) and a three-way junction point. (c) Red points indicate GFP-tubulin intensity peaks detected as anticlinal cortical microtubules. (d) Density of anticlinal cortical microtubules in interdigitation and three-way junction areas. Data are mean values from 134 and 161 independent regions, respectively. A statistically significant difference is observed using the Mann–Whitney U-test (p = 4.366e−8). (e) Relative strength of ROP2 (cyan) and ROP6 (magenta) activities in a numerical simulation. The three-way junction region functioned as a forced cytoplasmic protrusion at which the ROP2 pathway was expected to be dominant. ROP2 activity was monitored by the distribution of cortical microtubules.