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A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants

Hirokazu Tanaka¹, Hitoshi Onouchi^1,*, Maki Kondo², Ikuko Hara-Nishimura^2,, Mikio Nishimura², Chiyoko Machida^1, and Yasunori Machida^1,¶

¹ Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
² Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
^* Present address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Present address: Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Present address: Department of Biology, College of Bioscience and Biotechnology, Chubu University, Kasugai 487-8501, Japan

View larger version (73K): [in a new window]   Fig. 1. The phenotype of ale1 plants. (A-F) Gross morphology of aerial parts of plants. Plants were soil-grown in a covered container and the cover was removed 3 days after vernalization (DAV). (A) Wild-type plant, 7 DAV. (B,C) ale1-1 plants, 7 DAV. (D) Wild-type plant, 12 DAV. (E) ale1-1 plant, 12 DAV. (F) The same ale1-1 plant shown in E, at 16 DAV. The arrows in E and F indicate fused organs. (G,H) Transverse sections of wild-type (G) and ale1-1 (H) cotyledons. (I) Transverse section of fused leaves. (J,K) Adaxial surfaces of wild-type (J) and ale1-1 (K) cotyledons, 7 DAV. (L) Frequency of wrinkled leaves at each leaf position. Morphology of cotyledons and true leaves were observed 1 week and 3 week after vernalization, respectively. (M,N) Transmission electron micrographs of epidermal surfaces of wild-type (M) and ale1-1 (N) cotyledons. In the wild-type cotyledons, a densely staining layer covered the entire surface of each cotyledon. The arrowheads in M and N indicate the margin of the densely stained cuticle. cot, cotyledons; c, cuticle; cw, cell wall; cp, cytoplasm. Scale bars: 1 mm (A-F); 100 µm (G-I); 50 µm (J,K); and 1 µm (M,N).

View larger version (119K): [in a new window]   Fig. 2. Defects in ale1 mutants in cuticle formation and adhesion of the endosperm to the embryo’s surface. (A-E,K,M) Sections of wild-type seeds. (F-J,L,N) ale1-1 seeds. At each stage, seeds were sectioned and observed by light microscopy (A-J) or transmission electron microscopy (K-N). (A,F) Early heart-stage embryos. (B and G) Torpedo-stage embryos. (C,H) Magnified views of the boxed areas in B and G, respectively. (D,I) Walking-stick stage embryos. (E,J) Magnified views of the boxed areas in D and I, respectively. Arrows indicate adaxial surfaces of cotyledons. (K,L) Surfaces of embryos at the 16- to 32-cell embryo proper stage. The arrowheads indicate the thin electron-dense layer in the outermost region of the epidermal cell walls of the embryo. (M,N) Surfaces of heart-stage embryos. A continuous densely stained cuticle covered the entire wild-type embryo (M). The cuticle on the surface of the ale1-1 embryo was discontinuous (N; the arrowheads indicates the margin of the densely stained cuticle). em, embryo; en, endosperm; c, cuticle; cw, cell wall. Scale bars: 20 µm (A,B,F,G); 10 µm (C,E,H,J); 50 µm (D,I); 200 nm (K,L); and 500 nm (M,N).

View larger version (22K): [in a new window]   Fig. 3. Footprint alleles generated after excision of dAc-I-RS from the insertion allele ale1-1. The 8 bp duplicated upon dAc-I-RS insertion is labelled TSD. Footprints that leave the open reading frame intact (ale1-1-R1 to R4) lead to reversions to wild-type, while plants carrying the footprints that disrupt the open reading frame (ale1-1m1 and m2) are mutant.

View larger version (77K): [in a new window]   Fig. 4. Structure and patterns of expression of the ALE1 gene. (A) Schematic diagram of the ALE1 gene. White boxes, untranslated exons; gray boxes, translated regions. Positions of three amino acid residues (D, H and S) that are consistently conserved in the catalytic regions of the subtilisin-like serine proteases (the catalytic triad) are indicated (see B,C). The relative positions of the ale1 mutations are shown. (B) Predicted amino acid sequence of the ALE1 protein (accession number: AB060809). A putative signal peptide is underlined. Three residues in the catalytic triad are indicated by asterisks in B and C. (C) Alignments of amino acid sequences around the catalytic triad of subtilisin-like serine proteases. SDD1, A. thaliana; TPP2, human tripeptidyl peptidase 2; BPN’, Bacillus amyloliquefaciens, subtilisin BPN’; KEX2, Saccharomyces cerevisiae; FURIN, human; BLI-4, C. elegans (GenBank accession numbers: T00962, P29144, P00782, KXBY, P09958, and P51559, respectively). (D) The RNA on a gel blot prepared with poly(A)+ RNA from flowers, siliques at various developmental stages, and young plants (plantlets; 12 DAV) was allowed to hybridize with an ALE1 probe that covered part of the tenth exon and the 3' untranslated region. As a control, the washed membrane was subsequently hybridized with a probe for -tubulin (TUBA) (lower panel). S, Small siliques 2-4 days after pollination (DAP); M, medium siliques 3-6 DAP; L, large siliques 5-10 DAP.

View larger version (130K): [in a new window]   Fig. 5. ALE1 is expressed in endosperm surrounding developing embryos. (A-E) Pattern of distribution of ALE1 mRNA. (A) Seed containing the globular stage embryo. Signals (brown coloration) were observed in the endosperm around suspensor cells and weak signals were also detected in the embryo and suspensor cells themselves. (B) A globular-heart transition stage embryo and endosperm before cellularization. (C) Seed containing a heart-shaped embryo and cellularized endosperm. Strong signals (dark blue coloration) were observed in some of the cellularized endosperm that surrounded the embryo. (D) An embryo and endosperm at the heart stage. Strong signals were detected in some of the endosperm cells around the embryo. (E) Embryo and endosperm at the walking-stick stage. Signals were detected in some of the endosperm cells at positions closest to the embryo. (F,G) Sense controls for A to E. Reddish brown or weak blue signals in the cell layer underneath the seed coat and in the seed coat cells (indicated by white asterisks) were not specific to ALE1 RNA because they were generated by both the antisense and the sense probe. (H) GUS activity in endosperm cells of a transgenic plant that harbored the ALE1 promoter-GUS fusion gene. Scale bars, 50 µm.