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First published online 21 March 2007
doi: 10.1242/dev.003533

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Novel receptor-like kinase ALE2 controls shoot development by specifying epidermis in Arabidopsis

Hirokazu Tanaka^1,*, Masaru Watanabe², Michiko Sasabe², Tomonori Hiroe², Toshihiro Tanaka², Hirokazu Tsukaya³, Masaya Ikezaki², Chiyoko Machida¹ and Yasunori Machida^2,

¹ College of Bioscience and Biotechnology, Chubu University and CREST, Japan Science and Technology Agency, 1200 Matsumoto-cho, Kasugai 487-8501, Japan.
² Division of Biological Science, Nagoya University, Nagoya 464-8602, Japan.
³ Center for Integrative Bioscience, National Institute for Basic Biology, Okazaki 444-8585, Japan.

View larger version (130K): [in this window] [in a new window]   Fig. 1. The ale2 mutant is defective in surface functions and in the organization of epidermis-related tissues. (A,C) Gross morphology of 18-day-old plants. Arrows indicate fusion of leaves. (B,D) Defects on the surface of the epidermis, as revealed by the TB test. (E) Morphology of flowers and inflorescences of a wild-type plant (left; Col-0), an ale2-1 plant (middle) and an homozygous ale2-1 plant that harbored a 6.5 kb ALE2 transgene (right). (F,G) Scanning electron micrographs of mature wild-type (F) and ale2-1 (G) ovules. The ale2-1 ovules have a rough surface and have fused to one another. (H,I) Sections of mature wild-type (H) and ale2-1 (I) ovules. Asterisk indicates the embryo sac. i, integument; et, endothelium; mp, micropyle. Scale bars: 1 cm in D for A-D; 100 µm in F,G; 100 µm in I for I,H.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Map-based cloning and structure of the ALE2 gene. (A) Map position of the ALE2 gene on chromosome 2. The fractions shown indicate the number of recombinant chromosomes divided by the total number of chromosomes scored for each marker. The 55 kb region between the closest recombination points is indicated as a gray bar. (B) Structure of the ALE2 gene and the positions of mutations. Coding regions and untranslated regions are shown by black and white boxes, respectively. Introns are shown by horizontal bars. The length of each segment is indicated in bp. (C) Predicted sequence of the ALE2 protein. Two hydrophobic regions and a kinase domain are indicated by underlining and double underlining, respectively. Mutated amino acid residues in the ale2-1 allele and sites of T-DNA insertion in the ale2-2 allele relative to the wild-type amino acid sequence are indicated. (D) Comparison of the amino acid sequences of the putative extracellular region of ALE2 and the predicted sequences of other proteins. Clusters of basic amino acid residues are boxed. Positions of conserved cysteine residues are indicated by asterisks.

View larger version (38K): [in this window] [in a new window]   Fig. 3. ALE2 is an active protein kinase. (A) In vitro kinase activity of ALE2. GST-fusion proteins of the putative protein kinase domain of ALE2 (GST:ALE2KD) were tested to determine whether they have autophosphorylation activities (upper panel). Kinase activity of GST:ALE2KD was determined with MBP as a substrate (lower panel). (B) In vitro kinase activity of ACR4. The His-Nus-fusion protein of the ACR4 protein kinase domain autophosphorylated and phosphorylated MBP in vitro. (C) A mutual phosphorylation between ALE2 and ACR4. Effects of ALE2 or ACR4 on the activity of the other kinase were determined by kinase assays in vitro using an equal amount of recombinant ALE2KD and ACR4KD proteins. Four possible combinations of wild-type and kinase-inactive mutant proteins were examined. (D) Quantification of phosphorylation activities. The amounts of [-32P] ATP incorporated into the substrate proteins were determined. Wild-type ALE2KD and ACR4KD proteins increased the phosphorylation of kinase-inactive ACR4KD and ALE2KD, respectively (arrows). When either wild-type ALE2KD or ACR4KD protein was used as a substrate, a synergistic increase of phosphorylation by the other protein was observed (asterisks).

View larger version (66K): [in this window] [in a new window]   Fig. 4. The ale2 mutation affects fertility in a semidominant manner in the acr4 mutant background. (A) Inflorescence of self progeny obtained from an ale2-1/+; acr4-5/+ parent on the Ler background. Plants that were double homozygous for the ale2 and acr4 mutations were viable and resembled ale2 single-mutant plants. (B) Number of enlarged seeds per silique in the progeny of ale2-2/+; acr4-5/+ plants on the Ws background. Seeds from at least ten siliques were scored for each genotype. The genotype at each locus is indicated. Notice that, whereas ale2/+ plants were fully fertile (lanes 2 and 5) in the presence of the wild-type ACR4 allele, ale2 mutation affected fertility in a semi-dominant manner in the acr4 mutant background (indicated by the asterisk above lane 8; compare to lane 7). Essentially the same results were obtained for progeny of ale2-1/+; acr4-5/+ parents on the Ler background (data not shown). (C) Morphology of ovules. ale2 acr4 double mutants produced fused ovules that were similar to those of the ale2 single mutant. W, wild type; H, heterozygous; m, homozygous. Scale bars: 1 cm in A; 20 µm in C.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Synergistic effects of the ale1 and ale2 mutations on organ formation and on the formation of leaf cuticle. (A) Scheme for the isolation of ale1 ale2 double mutants. Approximately 25% of seeds from ale1: ale2/+ parents were either malformed (Aa) or shriveled (Ab). Seedlings from the malformed seeds had serious morphological defects, to various extents (Ac-Ae). (B) Epidermal surface defects verified with TB. Leaves of each single mutant were stained in a patchy pattern. Most of the surface of aerial parts of the ale1 ale2 double mutant was stained. (C) Quantification of the epidermal surface defects in 2-week-old plants by the TB test. (D) Transmission electron micrographs showing the surface of epidermal cells. Typical electrondense granules found in ale1 ale2 cells are indicated by asterisks. (E) Synergistic effects of the ale1 and ale2 mutations on shoot organization. Shoot apices of 5-day-old plants are shown. c, cuticle; cw, cell wall; cp, cytoplasm. Scale bars: 1 µm in D; 20 µm in E.

View larger version (79K): [in this window] [in a new window]   Fig. 6. Defects in cell morphology and organ formation during embryogenesis. (A-D) Differential-interference contrast images of cleared embryos of wild-type (Ler) (A), ale1-2 (B), ale2-1 (C) and ale1-2 ale2-1 double-mutant (D) embryos. Embryos are shown at the globular (G), the triangular (Tr), the heart (H) and the torpedo (T) stages. The three insets show gross morphology of respective embryos. (E) Frequencies of embryos with slightly swollen surfaces (white bars) and nearly spherical protodermal cells (black bars). The abnormal embryos from the indicated parents were scored. Scale bars: 20 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 7. Defects in ale1 ale2 and ale1 acr4 double mutants in the expression of protoderm-specific genes. (A-L) Accumulation of PDF1 (A-D), FDH (E,F,K,L) and ATML1 (G-J) transcripts, as visualized by in situ hybridization of the following embryos: wild type (A,E,G), ale1-1 (B), ale2-1 (C) and ale1-1 ale2-1 (D,F,H) on the Ler background; and wild type (I), acr4-1 (K) and ale1-1 acr4-1 (J,L) on the Ws background. (M-P) 5-day-old seedlings harboring the pPDF1::GFP construct. (M) A representative GFP-fluorescence image of a wild-type plant (inset) that expressed the pPDF1::GFP construct. (N) Magnified view of the central boxed region in M. (O,P) Gross morphology of an ale1 ale2 seedling with a disorganized leaf-like structure (O) and disorganized fluorescence due to GFP in the leaf-like organ (P). (P) Corresponds to the boxed area in O. The malformed young leaf exhibits a patchy pattern of fluorescence. Scale bars: 50 µm in A-L; 1 mm in M,O; 200 µm in N,P.

View larger version (30K): [in this window] [in a new window]   Fig. 8. A putative role for ALE2 in the promotion of protoderm differentiation. The RLK ALE2 and another RLK, ACR4, might function in a single or closely overlapping pathway. ALE1, encoding a putative subtilisin-like serine protease, is predominantly expressed in the endosperm that surrounds the developing embryo (Tanaka et al., 2001) and functions to promote the formation of the protoderm in a manner independent of ALE2 and ACR4. The two pathways involving ALE2, ACR4 and ALE1 might act positively to regulate the specification of the protoderm and/or expression of protoderm-specific genes in an organized manner. The expression of protoderm-specific genes, including those for the ATML1 homeodomain protein and for the redundant factor PDF2, might promote the expression of these genes themselves and other protoderm-specific genes (Abe et al., 2001; Abe et al., 2003). Subsequently, expression of the FDH gene for a putative fatty-acid elongase (Yephremov et al., 1999; Pruitt et al., 2000) and of other protoderm-specific genes promotes the formation of the epidermis proper.