Article Figures & Data
Figures
- Figure 1.
Rop2 Controls the Morphogenesis of Various Cell Types in Arabidopsis.
The effects of the transgenic expression of 35S:CA-rop2 and 35S:DN-rop2 fusion genes on cell shape in Arabidopsis plants were analyzed using different methods as described in the text. Images at left show wild-type plants, images in the middle show CA-rop2 transgenic plants, and images at right show DN-rop2 transgenic plants.
(A) to (C) Scanning electron microscopy images of leaf trichomes. Bar = 200 μm.
(D) to (F) Leaf cross-sections. Bar = 250 μm.
(G) to (I) Scanning electron microscopy images of leaf pavement cells. Bar = 20 μm.
(J) to (L) Pavement cells in cleared leaves. Bar = 40 μm.
(M) to (O) Hypocotyl cross-sections. Bar = 50 μm.
(P) to (R) Scanning electron microscopy images of hypocotyls. Bar = 100 μm.
- Figure 2.
CA-rop2 and DN-rop2 Expression Alters Radial Expansion but Not Longitudinal Expansion in Mature Pavement Cells.
(A) To determine whether Rop is involved in radial cell expansion, the average width of the neck region of wavy pavement cells was measured. Statistical analysis (t test) showed that pavement cells expressing DN-rop2 display significantly narrower necks than wild-type cells (n = 200; P ≤ 0.05).
(B) To determine whether Rop is involved in longitudinal cell expansion, the maximum length was measured from randomly chosen pavement cells of cleared leaves (see Figure 1). Statistical analysis (t test) showed no significant difference between wild-type cells and cells expressing CA-rop2 or DN-rop2 (n = 200, P > 0.05).
WT, wild type.
- Figure 3.
DN-rop2 and CA-rop2 Expression Does Not Alter the Length of Hypocotyl Epidermal Cells.
The average length of 150 epidermal cells located within 200 μm of the root-hypocotyl junction was determined from scanning electron microscopy images (see Figure 1). No significant difference was found between wild-type cells and cells expressing DN-rop2 or CA-rop2. WT, wild type.
- Figure 4.
Effect of CA-rop2 and DN-rop2 Expression on the Development of Different Stages of Leaf Epidermal Pavement Cells.
Expanding leaves (∼0.5 cm in length) from wild-type plants and transgenic plants expressing CA-rop2 or DN-rop2 were used for this analysis. Cell shapes were imaged by confocal microscopy using plants expressing the GFP-tubulin fusion gene as described in the text.
(A) to (C) Wild-type epidermal cells expressing GFP-tubulin.
(D) to (F) CA-rop2 epidermal cells expressing GFP-tubulin.
(G) to (I) DN-rop2 epidermal cells expressing GFP-tubulin.
(A), (D), and (G) show pavement cells in stage I; (B), (E), and (H) show pavement cells in stage II; and (C), (F), and (I) show pavement cells in stage III. WT, wild type. Bar in (A) = 20 μm for (A) to (I).
- Figure 5.
Distinct Effects of CA-rop2 and DN-rop2 Expression Distinguish the Rop-Dependent and Rop-Independent Phases of Anisotropic Cell Expansion.
(A) A “pseudo-time-course” analysis of radial and longitudinal expansion was conducted on hypocotyl cortex cells. The fifth cortex cells from three adjacent files were used in this analysis. Changes in cell expansion on these cortex cells were followed from days 1 to 5 after germination.
(B) and (C) Changes in average lengths and widths of hypocotyl cortex cells from days 1 to 5 after germination (n = 32). The cortex cells displayed their most rapid cell expansion in both the radial and longitudinal directions during days 2 and 3, although preferential expansion occurred in the longitudinal direction. Statistical analysis (t test) showed that during this period, longitudinal expansion was inhibited significantly by DN-rop2 expression, whereas radial expansion was promoted significantly by CA-rop2 expression (P ≤ 0.05). After day 3, only longitudinal expansion was observed, and neither radial nor longitudinal expansion was affected significantly by CA-rop2 or DN-rop2 expression.
WT, wild type.
- Figure 6.
Transition from Early to Late Phase of Cell Expansion Is Correlated with the Formation of Transverse Cortical MTs.
MT organization was analyzed in different stages of wild-type, DN-rop2, and CA-rop2 pavement cells using GFP-tagged α-tubulin as described in the text. In the early phase of cell expansion (between stages I and II), cortical MTs were oriented largely randomly, and DN-rop2 and CA-rop2 expression did not affect the organization of MTs. (A), (D), and (G) show late stage I cells from wild-type, DN-rop2, and CA-rop2 plants, respectively. During the transition from stage II to stage III, transverse MTs were found in certain regions of the cell in both wild-type plants (B) and DN-rop2 plants (H). CA-rop2 expression delayed the formation of transverse MTs slightly (E). Extensive transverse MTs remained even in fully expanded CA-rop2 cells (F). In contrast, in mature wild-type and DN-rop2 cells, cortical MTs were oriented randomly or longitudinally ([C] and [J]). All images shown except for (I) and (K) are projections of serial sections; (I) and (K) are medial sections showing cell shapes for (H) and (J), respectively. Bar in (A) = 20 μm for (A) to (K).
- Figure 7.
Formation of Diffuse Cortical F-Actin Is Associated with Cell Expansion and Is Altered by CA-rop2 and DN-rop2 Expression.
To visualize F-actin in live cells, GFP-mTalin was expressed transiently in leaf pavement cells of wild-type, CA-rop2, and DN-rop2 plants, and different stages of cells expressing GFP-mTalin were imaged using confocal microscopy as described in the text. All images shown are projections of serial laser sections except for (B′). Bar in (A) = 25 μm for (A) to (O).
(A) and (B) Wild-type stage I cells contained diffuse F-actin localized throughout the cell cortex.
(B′) Mid-plane section of the cell shown in (B) showing the cortical localization of diffuse actin and a cytoplasmic strand containing F-actin.
(C) A stage I cell showed no diffuse cortical actin when AtPFN1 is coexpressed with GFP-mTalin.
(D) A stage II cell contained strong diffuse F-actin in the cortical region of the lobe primordia.
(E) No diffuse cortical actin was found in a stage III cell, but an extensive network of actin cables was seen.
(F) to (J) CA-rop2 cells showed diffuse cortical actin at all stages: stage I (F), stage II ([G] to [I]), and stage III (J).
(K) Stage II CA-rop2 cell showed no diffuse cortical actin when AtPFN1 is coexpressed with GFP-mTalin.
(L) to (N) DN-rop2 cells contained little diffuse cortical actin in stages I ([L] and [M]) and II (N) and no diffuse cortical actin at stage III (O). Actin cables were present in all stages of pavement cells and were unaffected by CA-rop2 or DN-rop2 expression.
- Figure 8.
Transient Expression of CA-rop2 and DN-rop2 Generated an Opposite Effect on Cortical F-Actin.
GFP-mTalin was coexpressed with CA-rop2 or DN-rop2 in wild-type pavement cells and visualized by confocal microscopy as described for Figure 7 at two different times after particle bombardment (8 and 18 hr, respectively). Arrows point to loci of cortical F-actin at the first time point (8 hr after bombardment) and corresponding loci at the second time point (18 hr after bombardment). Two cells expressing GFP-mTalin alone showed no significant differences in F-actin distribution when visualized at 8 hr ([A] and [B]) and 18 hr ([A′] and [B′]), respectively. In cells expressing DN-rop2, localized diffuse cortical F-actin was reduced greatly at 18 hr ([C′] and [D′]) compared with 8 hr ([C] and [D]). On the contrary, CA-rop2 expression increased diffuse cortical F-actin dramatically during the same period ([E] versus [E′]). In contrast to the strong cortical F-actin in cells expressing CA-rop2 (F), no cortical F-actin was found in cells coexpressing AtPFN1 with CA-rop2, although normal actin cables were seen (G), showing that the cortical actin induced by transient expression of CA-rop2 was the same population of cortical F-actin induced by stable CA-rop2 expression, as shown in Figure 7. Bar in (A) = 15 μm for (A) to (G).
- Figure 9.
GFP-Tagged Rop2 Is Localized Preferentially to the Apparently Expanding Region of the PM.
To assess the subcellular localization of ROP2, GFP-ROP2 was expressed transiently in pavement cells and visualized by confocal microscopy. (A) and (B) show cells expressing GFP alone as a control. (C) to (I) show localization of GFP-ROP2. Arrows indicate strong GFP-ROP2 localization to specific cortical regions. Images shown are 1-μm laser sections. Bar in (A) = 20 μm for (A) to (I).
(A) and (B) Stage II and stage III cells, respectively. GFP was localized in the nucleus and throughout the cytoplasm.
(C) and (D) Two wild-type cells at stage I expressing GFP-ROP2.
(E) Two adjacent stage II cells expressing GFP-Rop2. Note a sinus region (arrowhead) in the bottom cell showing weak fluorescence, whereas its opposing lobe region in the neighboring cell showed strong PM fluorescence. Some regions of the cell were rich in cytoplasm, as indicated by the asterisk in the bottom cell, but contained weak fluorescence, indicating that the strong localized fluorescence was not attributable to dense cytoplasm in those regions.
(F) and (G) GFP-ROP2 localization in CA-rop2 pavement cells. GFP-ROP2 was distributed much more evenly throughout the PM in CA-rop2 cells at the equivalent stage (stage I) than in wild-type cells.
(H) and (I) PM localization of GFP-ROP2 in DN-rop2 pavement cells was very weak and was indistinguishable from its cytoplasmic localization.
