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First published online 1 November 2006
doi: 10.1242/dev.02684

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SUPPRESSOR OF FRI 4 encodes a nuclear-localized protein that is required for delayed flowering in winter-annual Arabidopsis

Department of Biology, Indiana University, Bloomington, IN 47405, USA.

View larger version (41K): [in a new window]   Fig. 1. SUF4 is required for the late-flowering phenotype of FRI and is alternatively spliced. (A,B) The effect of suf4 mutations on flowering time in the indicated genetic backgrounds. (A) Plants were photographed at similar stages of development (e.g. at the opening of the first flowers). (B) Bars represent the total number of leaves (rosette and cauline) formed by the primary shoot apical meristem. Black and gray bars represent plants grown under long days; white and cross-hatched bars represent plants grown under short days. Plants represented by gray and cross-hatched bars were cold-treated for 30 days before planting. Error bars represent the s.d. (C) Genomic structure of SUF4. Exons are indicated by open boxes; filled circles indicate sites of alternative splicing. (D) RT-PCR of SUF4 and a schematic drawing of the three splice variants of SUF4.

View larger version (55K): [in a new window]   Fig. 2. Alignment of SUF4 to related proteins. A putative nuclear localization signal is shown in bold (amino acid residues 3-7) and a region of high sequence identity between SUF4 and BAD46082 from rice is underlined. Proteins from C. briggsae, human, mouse, Drosophila and bee show significant sequence identity to the N-terminal part of SUF4 only; the C-terminal regions of these proteins are, therefore, not shown.

View larger version (59K): [in a new window]   Fig. 3. Spatial expression pattern of SUF4. (A) Nuclear localization of SUF4::GUS in trichomes. (B) DAPI-stained image of the same trichome used in A. (C,D) FLC::GUS expression and (E,F) SUF4::GUS expression in seedlings. Staining was performed 4 (C,E) and 10 (D,F) days after germination. SUF4::GUS expression in roots (G), the shoot apex (H), inflorescence (I) and developing seeds (J).

View larger version (31K): [in a new window]   Fig. 4. SUF4 expression, and its effect on flowering time and gene expression in FRI and AP-mutant backgrounds. (A) RT-PCR analysis of SUF4 in the early stages of development and in various tissues. Expression of SUF4 and FLC at 2-12 days after germination (DAG), and in shoots (S), roots (R), leaves (L) and flowers (F). (B) Effect of genotype and vernalization on SUF4 and FLC expression, as determined by RT-PCR. (C) RT-PCR analysis of FRI expression in wild-type (+) and suf4-mutant (-) backgrounds. (D) RT-PCR analysis of FLC expression in the indicated backgrounds with wild-type SUF4 (+) or mutant suf4 (-). (E) Effect of suf4 mutations on flowering time. Bars represent the total number of leaves (rosette and cauline) formed by the primary shoot apical meristem. Black bars represent the indicated genotypes with wild-type SUF4; white bars represent the indicated genotypes with the suf4 mutation. Error bars represent the s.d.

View larger version (29K): [in a new window]   Fig. 5. Effect of SUF4 on H3K4 trimethylation and gene expression at the FLC and FLM loci. (A) Schematic drawing of the FLC and FLM loci. White boxes represent the regions of FLC amplified in ChIP analysis (B and A), black boxes represent exons. (B) ChIP analysis of the histone H3-K4 trimethylation state of FLC chromatin in suf4 and related lines. The input is Col FRI chromatin before immunoprecipitation. `No AB' refers to the control sample lacking the anti-trimethyl H3-K4 antibody. `A' and `B' refer to the regions of FLC indicated in 5A. ACTIN served as an internal control. The results shown are representative of three replicates. (C) Effect of various mutations on the expression of FLC, FLM and neighboring genes, as determined by RT-PCR.