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Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3 – a katanin-p60 protein

Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK

View larger version (67K): [in a new window]   Fig. 1. erh3 mutants are short and hairy. Root phenotypes of 4-day old seedlings. Wild type (A,E), erh3-1 (B), erh3-2 (C,F), erh3-3 (D). Scale bar, 1000 µm (A-D), 250 µm (E,F).

View larger version (93K): [in a new window]   Fig. 2. Alignment of cell walls is defective in the meristems of erh3 mutant roots. Wild type (A,C,E), erh3-2 (B,D,F). Medial longitudinal sections (A,B). Transverse sections (C,D). Organisation of epidermal cells (E,F). a, atrichoblast (meristematic cell that will form a non-hair cell); t, trichoblast (meristematic cell that will form a hair cell). Scale bar, 100 µm.

View larger version (122K): [in a new window]   Fig. 3. Marker lines that are preferentially expressed in atrichoblasts, exhibit patchy expression in erh3-2 mutants, causing breakdown in ‘atrichoblast stripes’. GL2::GUS expression in wild-type (A) and erh3-2 mutant epidermis (B). Expression of the J2301::GFP enhancer trap in wild-type (C) and erh3-2 mutant (D) epidermis. Scale bar, 50 µm.

View larger version (76K): [in a new window]   Fig. 4. Double mutant phenotypes indicate that erh3 is epistatic to cpc and rhd6 for epidermal patterning and root-hair initiation. Scale bar, 500 µm.

View larger version (70K): [in a new window]   Fig. 5. Cells in the lateral root cap fail to express a lateral root cap marker in erh3 mutants. Expression of the J0481::GFP enhancer trap in wild-type (A,C) and erh3-2 mutant (B,D) meristems. (A,B) Projected images of GFP expression in the meristems. (C,D) Transverse sections approximately midway through the lateral root cap: top, PI-stained cell walls; bottom, GFP expression. Scale bar, 50 µm.

View larger version (120K): [in a new window]   Fig. 6. Cortical- and endodermal-specific marker is ectopically expressed in the epidermis of erh3 mutants. Expression of the J0571::GFP enhancer trap in wild-type (A,C) and erh3-2 mutant (B,D) meristems. (A,B) Medial longitudinal sections. (C,D) Transverse sections. Red arrowheads indicate ectopic expression in epidermal cells. Images of GFP expression were superimposed over images of PI stained cell walls. Scale bar, 50 µm.

View larger version (85K): [in a new window]   Fig. 7. A mature cortical cell-specific marker is ectopically expressed in the epidermis of erh3 mutants. Expression of the J0671::GFP enhancer trap in wild type (A,C) and erh3-2 mutants (B,D). (A,B) Longitudinal sections. (C,D) Transverse sections. Images of GFP expression were superimposed over images of PI stained cell walls. Scale bar 50 µm.

View larger version (30K): [in a new window]   Fig. 8. Mapping and organisation of the ERH3 gene encoding a p60 katanin-like protein. (A) ERH3 is located on the south of chromosome 1. Adjacent SSLP and CAPS markers, with the number of recombinant chromosomes identified, are indicated. The position of BAC clones spanning this region is indicated. The ERH3 gene is localised on BAC F5I6 (white box). The intron and exon organisation of the ERH3 gene, and the erh3-1, erh3-2 and erh3-3 mutations are shown. (B) Deduced amino acid sequence of the ERH3 gene product. The black box indicates the AAA domain. The mutated amino acids are indicated by asterisks. (C) Relationship analysis based on deduced amino acid sequence alignment of p60 katanin AAA domains using the neighbour-joining method (Thompson et al., 1997). The species origin of the sequence is indicated in brackets: Ce, C. elegans; At, A. thaliana; Cr, C. reinhardtii; Dm, D. melanogaster; Sp, S. purpuratus; Xl, X. laevis; Hs, H. sapiens; Mm, M. musculus. The GenBank accession number of the sequences are: At, AF048706; Ce,L25423; Cr, AF205377; Dm, AF223064, AE003601; Sp, AF052191; Xl, AF177942; Hs, AF056022; Mm, AF153197. Bootstrap values (100 replicates) are shown at the nodes. (D) Analysis of ERH3 expression by RT-PCR. Total RNA was isolated from leaves, flowers, roots and siliques of wild-type plants. A PCR reaction on a sample lacking the RT matrice was used as a negative control (Cont). Wild-type genomic DNA was used as a positive control.