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First published online 17 December 2003
doi: 10.1242/dev.00938

Development 131, 425-434 (2004)
Published by The Company of Biologists 2004
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RABBIT EARS, encoding a SUPERMAN-like zinc finger protein, regulates petal development in Arabidopsis thaliana

Seiji Takeda, Noritaka Matsumoto* and Kiyotaka Okada{dagger},{ddagger}

Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan



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  		Fig. 1. Structure of wild-type and rbe flowers. (A) A wild-type flower. (B,C) rbe-1 flowers. The arrowhead in B indicates the small petal, and black and white arrowheads in C indicate the spoon-shaped and filamentous petals, respectively. (D) An rbe-2 flower. Arrowheads indicate filamentous petals. Sepals have been removed in B-D. (E-I) Scanning electron micrographs of petals of wild type (E-G) and rbe-1 (H,I). (E) A stage 15 wild-type flower. (F,G) Epidermal cells on the upper (adaxial; F) and lower (abaxial; G) surfaces of a wild-type petal. (H) A rbe-1 filamentous petal. Note that it is formed at the normal position. The sepal on the right side has been removed. (I) A high magnification image of the filamentous petal in H. The epidermal cells are flattened and elliptical with irregular epicuticular ridges. (J) Diagram of a flower. Petals are numbered based on their position relative to the inflorescence meristem (IM). (K-N) Longitudinal sections of wild-type (K,M) and rbe-2 (L,N) flowers. (K) A wild-type stage 6 flower. Arrowheads indicate the petal primordia. (L) An rbe-2 stage 6 flower. Arrowhead indicates the position where petal primordia should form. (M) A wild-type stage 10 flower. Arrowheads indicate the elongated petals. (N) An rbe-2 stage 10 flower. Arrowheads indicate the positions where petal should form. se, sepal; p, petal; st, stamen; ca, carpel. Scale bars: E, 250 µm; F,G,I, 10 µm; H, 200 µm; K,L,M,N, 50 µm.

 


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  		Fig. 2. Scheme of map-based cloning and the structure of the RBE gene. (A) The genomic structure around RBE on chromosome 5. Numerals indicate the number of recombinants. Arrows indicate the open reading frames around the RBE gene. (B) cDNA and predicted amino acid sequences of RBE (GenBank accession no. AB107371; the locus name is At5g06070). The zinc finger motif is underlined, and the QALGGH motif specific to EPF zinc finger proteins in plants is boxed. The Potential nuclear localization signal is shown in bold type. The broken line indicates the leucine-rich domain of the carboxyl terminus. Mutations of rbe-1 and rbe-2 are shown. (C,D) Sequence alignment of the zinc finger domain (C) and the carboxy-terminal leucine-rich motif (D) in single zinc finger genes belonging to the RBE-SUP family in Arabidopsis thaliana. Asterisks in C indicate the conserved cysteine and histidine in the zinc finger domain. Consensus sequences are shown below the alignments.

 


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  		Fig. 3. An Arabidopsis thaliana suspension cell transiently expressing GFP protein (A-C) or RBE-GFP fusion protein (D-F). (A,D) Brightfield image; (B,E) DAPI (2 µg/ml) staining; (C,F) GFP fluorescence. Arrowheads mark the location of the nucleus. Scale bars: 100 µm.

 


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  		Fig. 4. RT-PCR analysis of RBE in tissues of the wild type. ACT8 was analyzed as a control. ST, stems; L, leaves; IM, inflorescence meristem, including young flower buds; OF, open flowers; SI, siliques; SE, seedlings; R, roots; G, genomic DNA for control.

 


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  		Fig. 5. Spatiotemporal expression patterns of RBE in inflorescences and floral buds. (A,B) RBE expression patterns in continuous transverse sections of a wild-type inflorescence. Arrowheads indicate RBE expression in stage 3 to 6 flowers. Asterisks indicate the position of the inflorescence meristem. (C,D) RBE expression patterns in longitudinal sections of a wild-type flower at stage 3 (C) and stage 6 (D). Arrowheads indicate RBE expression. (E) Top view of RBEp::GFP expression in a transgenic plants. Arrowheads indicate GFP expression. (F) RBEp::GUS expression in a transgenic plant. Arrowheads indicate GUS expression. Note that both RBEp::GFP and RBEp::GUS plants show the same expression patterns as with in situ hybridization. se, sepal; st, stamen; ca, carpel. Scale bars: 50 µm.

 


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  		Fig. 6. Structure of RBEp::DT-A flowers. (A-D) Scanning electron micrographs of wild-type (A,B) and RBEp::DT-A (C,D) flowers. (A) A wild-type stage 12 flower. (B) High magnification image of A. (C) An RBEp::DT-A stage 12 flower. (D) High magnification image of C. (E) A mature flower of the RBEp::DT-A plant. (F) High magnification image of E. Arrowheads in D and F indicate the position where petals should form. Sepals have been removed in A-D and F. p, petal. Scale bars: 100 µm.

 


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  		Fig. 7. Flower structure of rbe-1 ap3-5 double mutant and in situ localization of RBE transcripts in floral mutants. (A-D) SEM images of ap3-5 (A,B) and the rbe-1 ap3-5 double mutant (C,D). (B) A high magnification image of the second whorl organ in A. The stomata and long cells characteristic of sepal epidermis are present. (C) Arrowheads indicate underdeveloped organs formed in the second whorl. (D) A high magnification image of the second whorl organ in C. Stomata and long cells are present. (E,F) AP3 expression patterns in a transverse section of a wild-type inflorescence (E) and a longitudinal section of a stage 3 flower (F). (G,H) The AP3 expression patterns in a transverse section of a rbe-1 inflorescence (G) and a longitudinal section of a stage 3 flower (H). (I,J) RBE expression patterns in continuous transverse sections of an ap3-5 inflorescence (arrowheads). (K,L) RBE expression analysis in longitudinal sections of the early (K) and late (L) stage 3 flowers of 35S::PI 35S::AP3. RBE is not expressed in the organ primordia of the outermost whorl (shown as p'), but is expressed in the precursor cells of the second whorl organ primordia (arrowhead in L). (M,N) RBE expression in transverse sections of ag-1. Arrowheads indicate RBE expression. Numerals in N indicate the whorl number where the organ primordia form. (O,P) RBE expression analysis in a longitudinal section of ap1-1 (O) and a transverse section of ptl (P). RBE expression is not detected in the region where petal primordia would be expected to form (arrowheads). Asterisks in E, G, I, J, M and P indicate the position of the inflorescence meristem. se, sepal primordia; p', petal primordia in the outermost whorl; st, stamen primordia; ca, carpel primordia. Scale bars: A, 500 µm; B,D,E-P, 50 µm; C, 200 µm.

 

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© The Company of Biologists Ltd 2004