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First published online 19 July 2006
doi: 10.1242/dev.02508

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A role for chromatin remodeling in regulation of CUC gene expression in the Arabidopsis cotyledon boundary

¹ Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
² Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan.

View larger version (63K): [in a new window]   Fig. 1. atbrm mutants isolated in a genetic screen for phenotypic enhancers of cuc2-1. (A-F) 10-day-old cuc2-1 (A) or atbrm-1 (B) seedlings have well separated cotyledons. By contrast, cuc2-1 atbrm-1 double mutants exhibit cotyledon separation defects ranging from very strong (D) to moderate (E). cuc2-1 atbrm-3 also shows strong cotyledon separation defects (F). The strong cotyledon fusion (D,F) resembles that of cuc1-1 cuc2-1 double mutants (C). Genotypes are indicated in each panel. The cotyledons in cuc2-1 are labeled as `c' (A) to distinguish them from the primary leaves that form between the cotyledons. The outermost border of each fusion event is indicated by red arrowheads (D-F). Scale bars: 2 mm. (G) Mapping and cloning of the atbrm mutants. Asterisks indicate the position of CAPS markers used for fine mapping of the mutation in the H48 line. Arrows indicate predicted genes. The gene structure of AtBRM is represented by boxes for exons and by lines for untranslated regions and introns. (H) Molecular defects of the three atbrm alleles we identified. Structural domains of the protein are indicated: I, domain 1; II, domain 2; ATPase, SNF2 ATPase domain; purple oval, AT-hook; BM, bromodomain (e.g. Farrona et al., 2004).

View larger version (59K): [in a new window]   Fig. 2. Phenotypes of atbrm and syd. (A) atbrm-1 mutant (top) has a smaller rosette and curled leaves compared with the co-segregating wild-type plant (bottom). (B,C) Whereas the flowers in the wild-type inflorescence are completely open (C), flowers remain closed in the atbrm-1 inflorescence (B). (D,E) Fused stamen filaments (red arrowheads) can be found in the flowers of atbrm-1 (D) and syd-2 (E). (F) Root growth is retarded in atbrm-3 seedlings (left) relative to the wild type (right). Scale bars in B,C: 2.5 mm.

View larger version (45K): [in a new window]   Fig. 3. Phenotypic analyses of cuc and syd-2 or atbrm-1 double mutants. (A-C,E) Pictures taken at the indicated days after germination. None of the double mutants exhibit loss of shoot apical meristem function with the exception of cuc2-1 atbrm-1 (not shown); shoots form from the fused region of the cotyledons (yellow arrowheads in C-E). Genotypes are indicated in each panel. (D) Picture of a cleared seedling taken using DIC optics. Scale bars: 2 mm in A-C,E; 100 µm in D. (F) Percentage of plants showing cotyledon fusion in double mutant combination with cuc1-1, cuc2-1 or cuc3-101. Cup indicates cup-shaped seedling with substantial fusion on both sides of each cotyledon blade (as in B and E). CPF includes seedlings with complete fusion of one side of the cotyledon blade (see C), fusion of the cotyledon petiole, as well as cup-shaped seedlings. atbrm-1 mutants enhance all three cuc mutants, whereas syd-2 mutants only significantly enhance cuc1-1. The number of seedlings tested for each genotype is shown in parentheses.

View larger version (48K): [in a new window]   Fig. 4. Cotyledon fusion in double mutants between stm-2 and syd-2 or atbrm-1. (A) In stm-2, cotyledon fusion is negligible and the formation of one to two leaves (yellow arrowhead) can be observed at 10 days after germination (Clark et al., 1996). (B) Strong cotyledon fusion (petiole and base of cotyledons) was observed in stm-2 atbrm-1. (C) stm-1 exhibits only partial fusion of the cotyledon petiole (Barton and Poethig, 1993). (D) In stm-2 syd-2, cotyledon fusions are variable and range from frequent partial petiole fusion (red arrowhead) to stronger cotyledon fusions (heart shaped seedlings) (Kwon et al., 2005). Pictures were taken 10 days after germination. Scale bars: 2 mm. Genotype is indicated in each image. The outermost border of each fusion event is indicated by red arrowheads. (E) Real-time PCR evaluation of CUC message levels in single versus double mutants using biological duplicates. After normalizing with the message levels of the ubiquitously expressed translation initiation factor EIF4A, the resulting values for stm-2 were set to one to facilitate comparison between the lines. (F) Real-time PCR of ANT, FIL and UFO. The RNA analyzed was as in E. Shown are the mean and the s.e.m. of triplicate reactions normalized as in E. (G) Real-time PCR of CUC2 performed on RNA isolated from single mutant or wild-type inflorescences. Shown are the mean and s.e.m. of triplicate reactions normalized as in E.

View larger version (61K): [in a new window]   Fig. 5. CUC::GUS expression in embryos. (A) Top view of the embryo apex. The cotyledon boundary is indicated in turquoise: C, cotyledon; EM, embryonic meristem. (B-O) Siliques of cuc2-1/cuc2-1 atbrm-1/+ (B-I) and cuc1-1/cuc1-1 syd-2/+ (J-O) plants were examined. Genotypes are indicated to the right. cuc2 comprises cuc2-1/cuc2-1+/+ and cuc2-1/cuc2-1 atbrm-1/+; whereas cuc1 comprises cuc1-1/cuc1-1+/+ and cuc1-1/cuc1-1 syd-2/+. Representative embryos expressing CUC1::GUS (B-D,G,J,M), CUC2::GUS (E,H,K,N) and CUC3::GUS (F,I,L,O) are shown. When viewed from the front (f), CUC::GUS expression in the cotyledon boundary will appear as a small circle of expression (C-F,J-L). When viewed from the side (s) a line of expression will be observed (B). Embryos were at the torpedo stage (B,C) or at the bent cotyledon stage (D-O). Bent cotyledon stage embryos with fused cotyledons present a side view (s), whereas non-fused embryos usually present a front (f) view. Orientation (s,f) is indicated in each panel. In C, the same cuc2-1 atbrm-1 embryo is shown from two sides: nonfused (left) and fused (right). (G-I) CUC::GUS expression missing in the presumptive cotyledon boundary in the double mutants is indicated by a yellow arrow. (N) CUC2::GUS expression in the boundary between the cotyledons is severely reduced in cuc1-1 syd-2. Faint residual CUC2::GUS expression was observed in the presumptive boundary between each cotyledon and the shoot apical meristem, consistent with the observed lack of shoot apical meristem defects in cuc1-1 syd-2 (Fig. 2C). Where applicable, red arrowheads indicate the end points of cotyledon fusions (C,G-I,M-O). Scale bars: 100 µm.

View larger version (25K): [in a new window]   Fig. 6. A model for control of CUC gene expression in the embryo. (A,B) Diagrams indicating regulatory events in the cotyledon boundary region of an early heart stage embryo for CUC1 (A) and CUC2 (B). The arrows represent direct or indirect interactions. CUC1 and CUC2 are redundantly required to activate STM (Aida et al., 1999; Hibara et al., 2003; Takada et al., 2001). STM in turn is required for proper CUC1 and CUC2 expression (Aida et al., 1999; Aida et al., 2002; Kwon et al., 2005) (this study). AtBRM is a positive regulator of both CUC1 and CUC2, while SYD is required for CUC2 expression. Based on our phenotypical and molecular analyses, AtBRM and SYD are not likely to act in the STM pathway.

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