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. 2009 Feb;149(2):874-84.
doi: 10.1104/pp.108.132449. Epub 2008 Dec 12.

FZR2/CCS52A1 Expression Is a Determinant of Endoreduplication and Cell Expansion in Arabidopsis

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FZR2/CCS52A1 Expression Is a Determinant of Endoreduplication and Cell Expansion in Arabidopsis

Zachary Larson-Rabin et al. Plant Physiol. .
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Abstract

Endoreduplication, a modified cell cycle that allows cells to increase ploidy without subsequent cell division, is a key component of plant growth and development. In this work, we show that some, but not all, of the endoreduplication of Arabidopsis (Arabidopsis thaliana) is mediated by the expression of a WD40 gene, FIZZY-RELATED2 (FZR2). Loss-of-function alleles show reduced endoreduplication and reduced expansion in trichomes and other leaf cells. Misexpression of FZR2 is sufficient to drive ectopic or extra endoreduplication in leaves, roots, and flowers, leading to alteration of cell sizes and, sometimes, organ size and shape. Our data, which suggest that reduced cell size can be compensated by increased cell proliferation to allow normal leaf morphology, are discussed with respect to the so-called compensation mechanism of plant development.

Figures

Figure 1.
Figure 1.
Expression of FZR genes. A, GENEVESTIGATOR developmental summary of FZR expression data from microarrays. Tissues examined are: (1) germinated seed, (2) seedling, (3) young rosette, (4) developed rosette, (5) bolting plants, (6) young flower, (7) developed flower, (8) flowers and siliques, and (9) mature siliques. B, RT-PCR data from Arabidopsis tissues: Rs, rosette leaves; Fw, flowers; Cl, cauline leaves; Rt, roots; Sl, seedlings. C, qRT-PCR of FZR1, FZR2, and FZR3, normalized against UBIQUITIN10. Error bars indicate sd.
Figure 2.
Figure 2.
Expression and phenotypes of loss-of-function fzr2 mutations. A, Diagram of the FZR2 locus depicting exons (black boxes), the locations of the two T-DNA insertion mutations (white triangles), and the locations of primer sequences used in the RT-PCR analyses shown in B (arrows). B, RT-PCR analysis of mutants and FZR2 OE representatives. The FL bands resulted from RT-PCR using primers a and b, the 3′ segment used primers c and d, and the 5′ segment used primers e and f. C, Flowering wild-type (WT; left) and fzr2-1 (right) plants. D and E, ESEM micrographs of leaf trichomes of fzr2-1 (D) and wild-type (E) plants. Scale bar = 100 μm. F, Summary of leaf trichome branch production. Error bars represent ses. G and J, Representatives of DAPI-stained trichome nuclei of fzr2-1 (G and H) and wild type (I and J). The branch number for each trichome is given in the lower left of each picture. Scale bar = 15 μm. K, Summary of in situ fluorescence measurements of DAPI-stained trichome nuclei, given in relative fluorescence units (RFU).
Figure 3.
Figure 3.
Comparisons of cell area and endoreduplication profiles of wild type and fzr2-1. A, Histogram of epidermal pavement cell surface areas of wild-type (WT; average [ave.] = 23,570 μm2; sd = 18,560 μm2), fzr2-1 (ave. = 16,250 μm2; sd = 12,820 μm2), OE I (ave. = 153.5 μm2; sd = 109.6 μm2), and OE III (ave. = 22,480 μm2; sd = 13,935 μm2) plants. Cell surface areas were binned into 500-μm2 categories. B and C, ESEMs of adaxial leaf surfaces of wild type (B) and fzr2-1 (C). Scale bar = 100 μm. D and E, Flow cytometric (cyt.) profiles of PI-stained leaf nuclei of wild type (D) and fzr2-1 (E). A total of 10,000 events were recorded for each run. Ploidy and percentage of total nuclei are given at the top of each peak.
Figure 4.
Figure 4.
Phenotypes associated with FZR2 OE lines. A, Wild type (WT; right) and OE classes 28 d past germination. B, Relative expression of wild type and the three OE classes by qRT-PCR, normalized to UBIQUITIN10 expression. Error bars represent sds. C, ESEM micrographs of a leaf trichome of class OE I showing six branches. Scale bar = 100 μm. D to G, Representatives of DAPI-stained trichome nuclei of OE I trichomes. The branch number is given in the lower left of each picture. Scale bar = 15 μm. H and I, ESEMs of adaxial leaf surfaces of OE I (H) and OE III (I). Scale bar = 100 μm.
Figure 5.
Figure 5.
Cotyledon, root, stem, and guard-cell phenotypes of FZR2 OE lines. A and B, Cleared cotyledon of wild type (A) showing smaller mesophyll cells than those of an OE line (B). Black arrowheads indicate normal-sized mesophyll cells. Scale bar = 500 μm. C, ESEM of the adaxial surface of an OE I plant, showing both normal-sized (lower arrowhead) and enlarged (upper arrowhead) stomata. Scale bar = 50 μm. D to F, Light micrographs of DAPI-stained stomata of wild-type (D) and OE I plants (E and F). Scale bar = 10 μm. G, Root growth of wild-type (WT) and OE Class I seedlings after 9 d of growth on vertical 1.5% agar plates. H to M, Confocal images of PI-stained root tips of wild type (H), OE III (I), and OE I (J), and root mature zones of wild type (K), OE III (L), and OE I (M). Scale bar = 100 μm. N to P, DAPI-stained nuclei of root mature zones of wild type (N), OE III (O), and OE I (P). Q, Measurements of nuclear area of root cortex cells of wild-type and OE roots. Wild-type root cortex cell nuclei averaged 3,972 ± 1,179 μm2 (n = 46), OE III nuclei averaged 4,721 ± 1,529 μm2 (n = 22), and OE I nuclei averaged 12,090 ± 7,180 μm2 (n = 24). Student's t test (two-tailed with unequal variance) showed OE III to be marginally significantly different from wild type (P = 0.043), while OE I was very significantly different (P = 5.2 × 10−6). The OE I and OE III nuclei were also very significantly different from each other (P = 2.2 × 10−6). R and S, Cross sections of stems of wild-type (R) and OE III (S) plants. Black arrowheads indicate cortex tissue and black asterisks indicate pith tissue. Scale bar = 10 μm.
Figure 6.
Figure 6.
Phenotypes associated with wild-type and AP3FZR2 flowers. A, D, G, J, M, P, and S show floral tissue from wild type; B, E, H, K, N, Q, and T show AP3FZR2 weak phenotypes; C, F, I, L, O, R, and U show AP3FZR2 strong phenotypes. A to C, Inflorescences. D to F, Dissected petals. G to I, ESEM micrographs of petal epidermal cells. In I, white arrowheads indicate the multiple conical peaks of the giant cells. J to L, UV fluorescence micrographs of DAPI-stained petals. In L, a white arrowhead indicates a giant nucleus. M to O, Dissected stamens. P to R, ESEM micrographs of stamen filament. S to U, UV fluorescence micrographs of DAPI-stained stamens. Scale bar = 100 μm.

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