Article Figures & Data
Figures
- Figure 1.
Molecular Characterization of Selected AtADF1-O and AtADF-U Lines.
(A) RNA gel blot analysis. A total of 10 μg (top) or 30 μg (bottom) of total RNA prepared from 10-day-old seedlings was loaded in each lane. Blots were hybridized with antisense RNA transcribed from the AtADF1 5′ untranslated region and with a probe that recognizes 18S rRNA to control for loading. Closed arrows, endogenous AtADF1 mRNA; open arrow, transcript of the AtADF1 transgene (200 to 300 bp longer than the endogenous transcript because of the addition of the E9 poly[A] signal [Leu et al., 1996]). WT, wild type.
(B) Two-dimensional immunoblotting. For each panel, 80 μg of total protein was separated by SDS-PAGE followed by isoelectric focusing (IEF). A polyclonal antibody raised against AtADF1 was used to detect ADF proteins. Closed arrows, AtADF1; open arrows, other ADF isoform; arrowhead, spot detected exclusively when AtADF1-O protein preparations were blotted, likely representing phosphorylated AtADF1 (same molecular mass but different charge; AtADF proteins are known to be regulated by phosphorylation [Y. Hong and N.-H. Chua, unpublished data]).
- Figure 2.
Phenotypes of Light- and Dark-Grown Wild-Type, AtADF1-O, and AtADF-U Seedlings.
Seedlings grown in vitro for 10 days under a regular light regimen (left) or in complete darkness (right) are shown. Top row, wild-type (WT) seedlings; middle row, AtADF1-O seedlings; bottom row, AtADF-U seedlings.
. - Figure 3.
Elongation Kinetics of Etiolated Wild-Type, AtADF1-O, and AtADF-U Hypocotyls.
Seedlings were grown in vitro for 10 days in complete darkness. Each time point represents 15 seedlings. Open squares, wild type; open triangles, AtADF1-O; open circles, AtADF-U. Error bars indicate ±sd.
- Figure 4.
Scanning Electron Microscopy and Histological Analysis of Etiolated Hypocotyls.
Specimens were taken from the central region of the hypocotyls of 10-day-old dark-grown seedlings. (A) to (C) show surface views generated using scanning electron microscopy, and (D) to (I) show histological sections of cell walls stained with toluidine blue O. (D), (F), and (H) are cross-sections, and (E), (G), and (I) are longitudinal sections.
(A), (D), and (E) Wild type.
(B), (F), and (G) AtADF1-O.
(C), (H), and (I) AtADF-U.
Because of the wavy morphology of AtADF1-O hypocotyls, it was impossible to obtain longitudinal sections that showed extended stretches of vascular tissue. The vascular tissue in (G) therefore appears incomplete.
. - Figure 5.
F-Actin Organization in Epidermal Cells of Hypocotyls and Petioles.
(A) to (F) show GFP fluorescence emitted from the central regions of the hypocotyls of 10-day-old dark-grown (top row) and light-grown (middle row) GFP-mTn–expressing seedlings. (G) to (I) show fluorescein phalloidin fluorescence emitted from cotyledon petioles of 10-day-old dark-grown fixed seedlings. Projections of serial confocal optical sections are shown.
.(A), (D), and (G) Wild type (WT).
(B), (E), and (H) AtADF1-O.
(C), (F), and (I) AtADF-U.
- Figure 6.
Morphology and Actin Cytoskeleton of Specialized Cell Types.
(B), (D), (F), (H), (J), (L), and (N) show cell morphology as observed using scanning electron microscopy; (A), (C), (E), (G), (I), (K), and (M) show F-actin organization as visualized using stable expression of GFP-mTn. Projections of serial confocal optical sections are shown. All images depict selected cells with representative phenotypes. Wild-type trichomes display a variable degree of branching (Mathur and Chua, 2000), which was not affected significantly by AtADF1 overexpression (data not shown).
(A) and (B) Wild-type root hair.
(C) and (D) AtADF1-O root hair.
(E) and (F) AtADF-U root hair.
(G) and (H) Wild-type trichome.
(I) and (J) AtADF1-O trichome.
(K) and (L) Wild-type stomata.
(M) and (N) AtADF1-O stomata.
in (A) to (N) .
Tables
- Table 1.
Flowering Time (Days after Germination) and Rosette Leaf Number of Wild-Type, AtADF1-O, and AtADF-U Plants
Type of Plant Flowering Time Leaf Number Wild typea 35.33 (±1.377) 16.57 (±0.952) AtADF1-Oa 37.07 (±1.093) 16.46 (±1.098) AtADF-Ua 49.33 (±2.786) 25.61 (±1.903) a Data represent three independent experiments conducted with at least 15 plants of each genotype. The 95% confidence intervals are given in parentheses.
- Table 2.
Average Length (in millimeters) of Different Organs of 10-Day-Old Light-Grown Seedlings and of the Inflorescences of 68-Day-Old Adult Plants
Type of Plant Cotyledon Hypocotyl Root Inflorescence Wild typea 3.90 (±0.096) 2.59 (±0.096) 25.10 (±0.929) 288.5 (±11.08) AtADF1-Oa 2.13 (±0.088) 2.42 (±0.123) 21.45 (±0.386) 279.0 (±15.69) AtADF-Ua 4.28 (±0.123) 2.80 (±0.114) 27.75 (±0.815) 364.1 (±29.99) a The 95% confidence intervals are given in parentheses.
Type of Plant Root Hair Length (μm) Percentage of Wild Type Wild type 173.6 (±8.48) 100.0 AtADF1-Oa 128.3 (±4.97) 73.9 AtADF-Ua 225.0 (±9.45) 129.6 a Because the percentage of abnormal root hairs on individual AtADF1-O and AtADF-U plants was quite variable (see Discussion), transgenic plants with a high percentage (>70%) of affected root hairs were selected for this analysis. The 95% confidence interval is given in parentheses.
