The Putative Arabidopsis Arp2/3 Complex Controls Leaf Cell Morphogenesis

Characterization of the Arp2/3 subunit mutants. A, The location of the T-DNA insertions in arp2-1, arp2-2, arp3-1, and arpc5-1/p16-1 are shown on the maps of AtARP2, AtARP3, and AtARPC5/p16 genes. Arrows indicate the location of primers used for RT-PCR analysis of transcripts. Exons are boxed and introns and untranscribed flanking sequences are shown as lines. B, RT-PCR analysis of transcript levels for arp2-1, arp2-2, arp3-1, and arpc5/p16-1 mutants. Total RNA was extracted from inflorescences of each homozygous mutant plant and WT plants. The full-length coding sequence was amplified for each primer set.

Characterization of leaf pavement cell shapes in arp2-1 and arp3-1 plants. A, Leaf pavement cell shapes. The fourth rosette leaves from 2-week-old plants were cleared, and differential interference contrast images were taken from the middle region of the cleared leaves. Leaves from WT plants and all mutant plants are of similar sizes. Tracing was used to highlight the cell shape of a typical pavement cell. Bars = 30 μm. B, A schematic diagram of a leaf pavement cell illustrates how the lobe height and neck width were measured as shown in C and D. The distance indicated by dark arrows represents the lobe height. The distance indicated by light arrows represents neck width. C, Comparison of the average width of the neck region between WT and mutant pavement cells. Measurements in both C and D were carried out using the fourth leaf of a 2-week-old plant, and 150 epidermal cells were used for each mutant line as well as WT. Statistical test (t test) shows no significant difference in neck width between WT and mutant pavement cells. D, Comparison of the average length of lobe heights between WT and mutant pavement cells. Statistical test (t test) shows that the length of lobe height is significantly reduced in the mutant pavement cell compared with the WT pavement cell.

Trichome phenotypes of arp2-1, arp3-1, and arpc5-1 mutants. A, Phenotype analyses of arp2-1, arp3-1, and arpc5-1 mutant trichomes using light microscopy. a to d, Trichomes on a young leaf from WT and mutant plants. e to h, Single trichomes with branches from WT and mutant plants. Arrows point to the branches. i to l, Trichomes on stems from WT and mutant plants. a, e, and i are from WT plants. b, f, and j are from arp2-1 plants. c, g, and k are from arp3-1 plants. d, h, and l are from arpc5-1 plants. Arrows in e to h point to the branches. Bar in a (100 μm) is the same for b through d. Bar in e (50 μm) is the same for f through h. Bar in i (500 μm) is the same for j through l. B, Scanning electron microscopic analyses of arp2-1 trichomes. a and b show mature trichomes on a young leaf from WT and arp2-1 plants, respectively. c and d show single trichomes with the typical three branches from WT and arp2-1 plants, respectively. Bar in a and b = 500 μm. Bar in c = 100 μm. Bar in d = 20 μm.

Analysis of actin organization in leaf pavement cells of arp2-1 and arp3-1 plants. A, F-actin organization in leaf pavement cells from arp2-1 and arp3-1 plants visualized by GFP-mTalin at different developmental stages. Leaves from 10-d-old plants were bombarded with GFP-mTalin construct, and cells expressing GFP-mTalin were imaged using confocal microscopy as described in the text. Pavement cells at three developmental stages are shown: b, g, and k, Stage I cells before lobe initiation; c, h, and m, stage II cells forming lobes; and e, j, and o, stage III cells with fully extended lobes. More than 60 cells were imaged for each stage of cell in every genotype. Shown are cells with representative actin organization. a to e, Leaf pavement cells of WT plants. f to j, Leaf epidermal cells of arp3-1 plants. k to o, Leaf epidermal cells of arp2-1 plants. a, c, e, f, h, j, k, m, and o show three-dimensional images projected from laser scanning. b, d, g, i, l, and n are single medial sections of a, c, f, h, k, and m, respectively. Arrows in c, h, and m point to the diffuse F-actin patches associated with growing lobes. Arrowheads in h and m point to diffuse F-actin associated with neck regions. Asterisks in h and i indicate the nucleus of the cell. Bar = 15 μm for all images. B, Quantitative analysis of association of diffuse cortical F-actin with the tip of expanding lobes in pavement cells. Measurements were done on 30 stage II cells by visual examination. The percentage of strong diffuse cortical F-actin associated with lobes for WT, arp2-1, and arp3-1 pavement cells are 70.83 ± 10.57, 50.22 ± 12.08, and 47.08 ± 10.91, respectively. Statistical analyses (t test) showed that the percentage of strong diffuse F-actin associated with lobes of both mutant pavement cells is significantly reduced (n = 30, P ≤ 0.05).