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SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG

¹ Department of Cell Biology and Molecular Genetics, 3236 H.J. Patterson Hall, University of Maryland, College Park, MD 20742, USA
² Syngenta, 3054 Cornwallis Road, Research Triangle Park, NC 27709, USA

View larger version (106K): [in a new window]   Fig. 1. Phenotypes of seu mutant plants. A-C,I are photographs; D-H and J-K are scanning electron micrographs. (A) A wild-type flower. (B) A seu-1 mutant flower with narrow petals (arrow) and narrow sepals (arrowhead). (C) A seu-2 mutant flower exhibiting a whorl 1 stamen/carpel mosaic organ (arrowhead) and a whorl 2 petal that is reduced in size and is staminoid (arrow). The tip of the gynoecium is unfused, exposing the ovules (o). (D) Abaxial surface of a seu-1 whorl 1 organ. Both petal (pe) and sepal (se) cells are present. (E) An enlargement from the boxed area in D. Both round-shaped petal cells (arrow) and rectangular-shaped sepal cells (arrowhead) are present. (F) Wild-type petal cells. (G) Wild-type sepal cells. (H) A seu-2 mutant flower. Note the long tubular organs (arrows) in whorl 2. These tubular organs are likely petal in identity because of the petal blade at the tip of the tube. (I) Wild-type and mutant plants. All plants are Ler ecotype. The ruler is 15 cm long. The seu-1 plant is about half the height of the wild-type one; seu-1 ap2-1 is similar in height to seu-1; seu-1 lug-8 double mutant is less than half the height of seu-1 and is bushy. Inset box is an enlarged picture of a seu-1 lug-8 plant. (J) A mature wild-type ovule. (K) A seu-1 ovule with an abnormal protrusion at the micropylar end (arrowhead). Scale bars in D, E, G, J, K are 100 µm; bar in F is 50 µm; bar in H is 1 mm.

View larger version (149K): [in a new window]   Fig. 2. seu-1 enhances the floral defects of lug and ap2. A-B,E-F,J are photographs; C-D,G-I,K-L are SEMs. (A) A lug-1 mutant flower with narrow petals (arrow) and split gynoecium (arrowhead). (B) A seu-1 lug-1 double mutant flower at twice the magnification of the flower in A. The seu-1 lug-1 flower is roughly 25% size of a wild-type flower. Only two carpelloid organs with horns (h) are formed in whorl 1. A single stamen forms in whorl 3. Whorl 4 is reduced to a small mound of tissue (arrowhead). (C) A seu-1 lug-2 flower. Whorl 1 organs are carpelloid (arrow) but lack stigmatic tissue and ovules. Whorl 4 is just a small mound of tissue (arrowhead). (D) A seu-1 lug-8 mutant flower. The small mound of tissue in whorl 4 (arrowhead) forms two ovule primordia (op). Inset box is an enlargement of the ovule primordia. (E) An ap2-1 mutant flower. Whorl 1 organs are leaf-like with trichomes characteristic of leaves (arrow). (F) A seu-1 ap2-1 double mutant flower. Whorl 1 organs are carpelloid with stigmatic tissues (arrowhead) and ovule primordia (op). (G) An ap2-2 flower. The relatively normal whorl 4 gynoecium is indicated (arrow). Whorl 1 organs are carpelloid with stigmatic tissus (arrowhead) and ovule primordia (op). (H) A seu-1 ap2-2 double mutant flower. Whorl 1 organs are carpelloid with ovule primordia (op) along their margin but lack stigmatic tissues. A stamen/sepal mosaic organ was removed (arrow) to reveal whorl 4 that is reduced to a mound of tissue (arrowhead). (I) A seu-1 flower. Two whorl 1 sepals have been removed to reveal a narrow petal (arrow) and an unfused gynoecium (arrowhead). (J) A seu-1/seu-1; ap2-2/+ mutant flower. The presence of ap2-2/+ enhances seu-1/seu-1 as shown by a stamenoid/carpelloid organ (arrowhead) in whorl 1 and a stamenoid petal (arrow) in whorl 2. (K) A seu-1/seu-1; lug-1 + /+ ap2-1 flower. The presence of lug-1 + /+ ap2-1 further enhances seu-1/seu-1. A whorl 1 organ with both stamen and carpel characteristics is indicated (arrowhead). (L) A seu-1/seu-1; lug-1 +/ + ap2-1 flower. Note the carpelloid whorl 1 organ with ovule primordia (op) on the organ margin and a horn (h) on the top. Scale bars in C, D, I, K, and L, 1 mm; in G and H, 100 µm.

View larger version (94K): [in a new window]   Fig. 3. Ectopic and precocious expression of AG in seu single and seu lug double mutants. 8 µm longitudinal sections of Arabidopsis inflorescences were hybridized with an AG antisense probe. Numbers indicate stages of floral development [based on Smyth et al. (Smyth et al., 1990)]. (A) A wild-type inflorescence. AG mRNA is detected in the center of the stage 5 flower and is not detected in sepal primordium (arrowhead). AG mRNA is not detected in stage 2 floral meristems. (B) A seu-1 inflorescence. AG mRNA is detected in the stage 2 floral meristem and in the sepal primordia of stage 3 floral meristem (arrowhead). (C) A seu-1 lug-8 double mutant inflorescence. AG mRNA is detected as early as the stage 1 and stage 2 floral meristems. Expression of AG mRNA is also detected in groups of cells (marked with a *) in the inflorescence meristem that might represent pre-stage 1 cells. AG mRNA is detected in the sepals of all flowers (arrowheads). Note the severely reduced whorl 4 (white arrows). The pink color in the stem is residual Eosin stain and does not reflect hybridization signal.

View larger version (153K): [in a new window]   Fig. 4. SEMs of seu-1 ag-1 and seu-1 lug-1 ag-1 flowers. Number indicates whorl number. (A) An ag-1 flower. Whorl 1 organs are sepals, and whorls 2 and 3 organs are petals. Whorl 4 is another flower repeating the (sepal-petal-petal)n pattern. (B) A seu-1 ag-1 double mutant flower. Whorl 1 organs are narrow sepals. Whorls 2 and 3 are narrow petals. Whorl 4 is another flower. (C) A seu-1 lug-1 ag-1 triple mutant flower. The whorl 1 sepals are very narrow and canoe-shaped. Whorls 2 and 3 organs are either blade-like or club-like (arrowhead). (D) A close-up image of the epidermal cells in the blade-like organ. These epidermal cells exhibit characteristics of petal cells. (E) A close-up image showing several developing blade-like organs (arrowhead) in whorls 2 and 3 of a seu-1 lug-1 ag-1 triple mutant flower. (F) A close-up image showing several club-like organs (arrowhead) in whorls 2 and 3 of a seu-1 lug-1 ag-1 triple mutant flower. Scale bars in A, B, and C are 1 mm; scale bar in D is 10 µm; scale bars in E and F are 100 µm.

View larger version (71K): [in a new window]   Fig. 5. Molecular cloning of the SEUSS gene. (A) A physical map of the SEU region on Arabidopsis chromosome I. Percentage recombination for five CAPS markers is shown. n indicates the number of meiotic products examined at the given marker. BAC clones are shown as open boxes. The SEU gene, shown as a shaded box, maps 0.16 cM north of the CAPS marker F9L16Sp6. (B) Nucleotide sequence and the predicted amino acid sequence of SEU. Numbers on the right indicate the amino acid residue. The boxed area encodes a bi-partite nuclear localization signal. The underlined sequence is the putative dimerization domain with similarity to the Ldb proteins. The filled triangles indicate seu-1 and seu-2 mutations. The seu-1 allele is caused by a C to T transition, resulting in a ‘TAA’ stop codon at amino acid 501. The seu-2 allele is caused by a single base-pair deletion of the ‘G’ base indicated. The full length cDNA sequence (3555 bp) including 5' and 3' UTR has been deposited with GenBank (AF378782).

View larger version (24K): [in a new window]   Fig. 6. SEU mRNA expression. (A) Northern analysis of mRNA isolated from flowers of wild-type and seu mutants. The doublet bands of SEU mRNA reflect two different transcriptional initiation sites confirmed by 5'RACE (see text). The relative mRNA level is corrected with the 18S RNA as a loading control and compared with wild-type signal level. (B) Northern analysis of mRNA isolated from leaves and flowers of wild-type plants. SEU mRNA is expressed at a higher level in flowers than in leaves.

View larger version (75K): [in a new window]   Fig. 7. Sequence similarity between SEU, SEUSS-LIKE proteins and Ldb proteins. (A) Diagramatic representation of the open reading frames of SEU, SEUSS-LIKE proteins from Oleracea sativa (AAF34437) and A. thaliana (CAA18174), representative Ldb family members from Danio rerio (AF031377), and Mus musculus (AF024524) and a putative Ldb family member from Saccharomyces pombe (AL031262). Numbers indicate amino acid residues. The shaded portion represents the putative dimerization domain for each protein. Percentages shown within the putative dimerization domain indicate amino acid sequence identity between SEU and the respective protein. Percentages shown to the right are percentage identity to SEU over the entire open reading frame. Glutamine-rich regions are shown as hatched boxes. LID: LIM interaction domain. (B) Sequence alignment of SEU with representative SEUSS-LIKE proteins, and Ldb family proteins in the putative dimerization domain. Identical residues are shaded black. Similar residues are shaded gray. The predicted alpha-helical portion of SEU is indicated with a two-headed arrow. Four hydrophobic or non-polar amino acids (•) in SEU are spaced seven residues apart in this region. Six hydrophobic or non-polar amino acids (•) in the M. musculus Ldb1 protein are also spaced seven residues apart, suggesting a hydrophobic zipper structure (Jurata and Gill, 1997).