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First published online 14 February 2007
doi: 10.1242/dev.02803

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Transcriptional regulation of epidermal cell fate in the Arabidopsis embryo

Developmental Genetics, Center for Molecular Biology of Plants, University of Tübingen, D-72076 Tübingen, Germany.

View larger version (103K): [in this window] [in a new window]   Fig. 1. ATML1 promoter activity in wild-type and mutant embryos. (A, B) ATML1 promoter-GFP reporter fusion constructs pATML1::NLS:3xEGFP (A) and pATML1::CYCB1-N:NLS:3xEGFP (B). NLS, SV40 nuclear localization signal sequence; GFP, enhanced green fluorescence protein sequence; nost, nopaline synthase terminator; CYCB1-N, a sequence encoding an N-terminal fragment of CYCLINB1;2, including the destruction box. (C-H) pATML1::NLS:3xEGFP expression in wild-type embryos at successively older stages: (C) one-cell; (D) 16-cell; (E) 32-cell; (F) mid-heart (arrowheads indicate quiescent center cells lacking GFP signal); (G) torpedo; (H) bent-cotyledon. (I,J) pATML1::CYCB1-N:NLS:3xEGFP expression in wild-type embryos at different stages: (I) two-cell (arrowhead indicates GFP expression in nucleus of suspensor cell), and (J) 16-cell (compare with D). (K-N) pATML1::NLS:3xEGFP expression in mutant embryos: (K) ball-shaped gn; (L) heart-stage mp; (M) basal peg of mp; (N) atml1;pdf2. Green, GFP signals; red, chlorophyll autofluorescence. Scale bars: 10 µm in C-G,I-N; 50 µm in H.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Expression analysis of ATML1 promoter regions. Deletions of promoter regions A, B, C and D are illustrated to the left; corresponding GFP expression patterns at the one-cell stage (early embryo) and heart stage (epidermis) are shown to the right. Presence or absence of GFP expression (green or yellow) are indicated by + or -, respectively. *1, one of nine lines showed expression at the one-cell stage; *2, no GFP expression in the apical half of the embryo proper; *3, GFP expression only in the central region of the embryo. In the schematic, the arrow (top diagram) indicates the transcription start site (+1); 35Smini, minimal promoter derived from the cauliflower mosaic virus 35S promoter. Scale bars: 10 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 3. Position-dependent GFP expression along the apical-basal axis conferred by ATML1 promoter deletions. (A) `Basal half' expression at the 32-cell stage in A. (B) `Apical lineage' expression at the early-heart stage in AB. (C-F) GFP expression in ABC at the eight-cell (C), 32-cell (D), mid-heart (E) and late-heart (F) stages. (G) `Basal lineage' expression at the eight-cell stage in AD. (H) `Apical lineage' expression at the 16-cell stage in B. (I) `Central' expression at the late-heart stage in BCD. (J-L) GFP expression in D at eight-cell (J), 32-cell (K) and late-heart (L) stages. Scale bars: 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Effects of small deletions within region D on ATML1 promoter activity. To the right of each deletion construct, presence or absence of promoter activity at the one-cell stage (early embryo) and heart stage (epidermis) are indicated by + or -, respectively. 35Smini, 35S minimal promoter; n.d., not done; *1, only six of 20 transformants showed expression; *2, only four of 22 transformants showed expression. Scale bars: 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 5. GFP expression driven by six tandem repeats of the 179 bp promoter fragment. The diagrams to the left indicate the position of the 179 bp fragment in the ATML1 promoter, the WUS-binding site (red circle) and the L1 box (yellow circle). The arrow indicates the transcription start site. Red crosses indicate mutations in cis-regulatory sequences. All fragments consisted of six tandem copies (x 6) fused to the 35S minimal promoter (35Smini). (A-E) 6x179bp expression at one-cell (A), eight-cell (B), 32-cell (C), early-heart (D) and late-heart (E) stages. (F-J) 6x179bpmL1 expression at one-cell (F), eight-cell (G), late-globular (H), early-heart (I) and late-heart (J) stages. (K-O) 6x179bpmWUS expression at one-cell (K), eight-cell (L), 32-cell (M), early-heart (N) and late-heart (O) stages. Scale bars: 10 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 6. GFP expression driven by four overlapping subfragments of the 179 bp promoter fragment. The constructs are shown to the left, the corresponding GFP expression patterns in embryos to the right. All the promoter subfragments were hexamerized (x 6) and fused to the 35S minimal promoter (35Smini). Sequence numbering refers to the distance in bp from the transcription start site (+1; arrow in top diagram). Circles indicate the WUS-binding site (red) and L1 box (yellow). GFP expression in (A-E) 6x89bp, (F-J) 6x101bp, (K-O) 6x114bp, (P-T) 6x90bp. Embryo stages: (A,F,K,P) one-cell; (B,G,L,Q) eight-cell; (C,H,M,R) 32-cell; (D,I,N,S) mid-heart; (E,J,O,T) late-heart. White arrowheads (D,E,N) indicate reduced expression in the presumptive SAM region; yellow arrowheads (C-E,M) indicate reduced expression at the basal pole; white arrows (S) indicate patchy expression in epidermis. Scale bars: 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Model for the transcriptional regulation of ATML1 in embryogenesis. (A) Regulatory organization of the ATML1 promoter. Target regions of putative positional signals (arrows) are indicated. Sequence numbering refers to the distance in bp from the transcription start site (+1). (B) Regulation of ATML1 expression in embryogenesis. Each color represents a different expression domain. The regulatory sequences that mainly contribute to the expression in each domain are indicated (see text for details). Successive embryo stages are depicted (from left to right): one-cell stage, eight-cell stage, globular stages, heart stage.