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Research ArticleCELL BIOLOGY AND SIGNAL TRANSDUCTION
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The Putative Arabidopsis Arp2/3 Complex Controls Leaf Cell Morphogenesis

Shundai Li, Laurent Blanchoin, Zhenbiao Yang, Elizabeth M. Lord
Shundai Li
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Laurent Blanchoin
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Zhenbiao Yang
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Elizabeth M. Lord
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Published August 2003. DOI: https://doi.org/10.1104/pp.103.028563

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    Figure 1.

    RT-PCR analysis of the transcript expression of the Arabidopsis Arp2/3 subunit genes in various tissues. RNA was isolated from organs of Arabidopsis COL-0 WT plants grown in light and used for RT-PCR analysis as described in the text. A 25-cycle PCR reaction was carried out for all genes except for ARPC4/p20, for which 30 cycles were used. ACTIN2 was used as a control except for pollen RT-PCR that involves the use of the ACTIN3 gene.

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    Figure 2.

    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.

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    Figure 3.

    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.

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    Figure 4.

    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.

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    Figure 5.

    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).

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    Figure 6.

    Actin organization in trichomes of arp3-1 and arp2-1 plants. F-actin was visualized as described in Figure 5. All images shown are projections. Bar in A is for A, D, and G. Bar in B is for B, E, and H. Bar in C is for C, F, and I. Bar = 20 μm. A, D, and G, Young trichomes from a WT, arp2-1, and arp3-1 plants, respectively. Arrow in A points to diffuse cortical F-actin signal in a rapid growing branch. Arrows in D and G indicate the loss of fine F-actin in the mutant branches. Arrowheads in D and G point to thick actin cables. B, E, and H, Stalks of mature trichomes from WT, arp2-1, and arp3-1 plants, respectively. C, F, and I, Branches of mature trichomes from WT, arp2-1, and arp3-1 plant, respectively.

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    Table I.

    Comparison of Arabidopsis Arp2/3 complex subunits with their counterparts in other organisms

    Human Fruitfly Dictyostelium sp. Fission Yeast Budding Yeast
    ARP3 56a 55 57 53 51
    ARPC1/p41 41 37 43 35 33
    ARPC2/p34 26 26 33 26 28
    ARPC3/p21 47 42 41 43 39
    ARPC4/p20 65 61 68 56 59
    ARPC5/p16 32 35 31 25 23
    • ↵a The number indicates percentage of identity of predicted amino acid sequences of Arabidopsis Arp2/3 subunits compared with those from other species

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The Putative Arabidopsis Arp2/3 Complex Controls Leaf Cell Morphogenesis
Shundai Li, Laurent Blanchoin, Zhenbiao Yang, Elizabeth M. Lord
Plant Physiology Aug 2003, 132 (4) 2034-2044; DOI: 10.1104/pp.103.028563

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The Putative Arabidopsis Arp2/3 Complex Controls Leaf Cell Morphogenesis
Shundai Li, Laurent Blanchoin, Zhenbiao Yang, Elizabeth M. Lord
Plant Physiology Aug 2003, 132 (4) 2034-2044; DOI: 10.1104/pp.103.028563
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Plant Physiology: 132 (4)
Plant Physiology
Vol. 132, Issue 4
Aug 2003
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