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First published online 15 September 2004
doi: 10.1242/dev.01363

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The Arabidopsis thaliana SNF2 homolog AtBRM controls shoot development and flowering

¹ Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Américo Vespucio s/n, E-41092 Sevilla, Spain
² Section of Plant Biology, University of California, Davis, Davis, CA 95616, USA

View larger version (19K): [in a new window]   Fig. 1. Domain organization of proteins of the SWI2/SNF2 subfamily in Arabidopsis. The domains distribution of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila Brahma proteins are also included. Numbers correspond to amino acid positions. The black bar above AtBRM indicates the region encoded by the DNA fragment used for RNA interference.

View larger version (22K): [in a new window]   Fig. 2. AtBRM is a nuclear protein associated with a high molecular mass complex. (A) Nuclear and cytoplasmic proteins from inflorescence apices were subjected to western blotting using two different antisera against two different regions of AtBRM (-AtBRMa and -AtBRMb). (B) Gel filtration analysis of nuclear extracts of Arabidopsis calli. Fractions from chromatography through a Superose 6 HR column were separated by SDS-PAGE and examined by western blotting using -AtBRMb antiserum. Fraction numbers are given across the top and arrows indicate elution position of molecular mass standards.

View larger version (70K): [in a new window]   Fig. 3. AtBRM expression pattern. (A) AtBRM mRNA expression in different tissues. RNA was isolated from adult plants grown in long days. Total RNA blots were hybridized first with an AtBRM probe and then reprobed with 18S ribosomal DNA (18S) as a loading control. Level of AtBRM mRNA was also determined by RT-PCR (bottom panel). (B) AtBRM protein expression in different tissues. 10 µg of nuclear protein were separated by SDS-PAGE, and subjected to western blotting using -AtBRMb antiserum. (C-H) Histochemical GUS staining of transgenic Arabidopsis plants containing an AtBRM promoter-first intron-GUS fusion (GUS staining is blue in whole mount, and pink when viewed with dark-field optics). (C) Ten-day-old whole seedling. Bar, 1 mm. (D) Magnification of a young leaf, showing staining of the vascular tissue. Bar, 1 mm. (E) Magnification of root tips of seedlings. Scale bar: 0.1 mm. (F) Inflorescence meristem. Scale bar: 0.1 mm. (G) Cross section of a vegetative shoot apical meristem, showing first leaves, leaf primordia and cotyledons. (H) Longitudinal section of floral bud at stage 12. Scale bar: 0.25 mm.

View larger version (71K): [in a new window]   Fig. 4. Phenotypes of AtBRM-silenced plants (atbrm). (A) Levels of AtBRM mRNA in different kanamycin-resistant transgenic lines transformed with pART27-AtBRM-RNAi. Total RNA was isolated from inflorescence apices, blotted and hybridized with an AtBRM probe. The blot was stripped and rehybridized with an 18S ribosomal probe (18S) as a loading control. (B) Levels of AtBRM protein in AtBRM-silenced plants. 20 µg of nuclear protein were subjected to western blotting using -AtBRMb antiserum. (C) Wild-type (Columbia) and atbrm (line 29.1) plants at 23 days of growth in LD. (D) Higher magnification of one atbrm plant shown in C. Scale bar: 5 mm. (E) Rosette leaves from wild-type and atbrm (lines 10.1 and 29.1) plants grown in LD. Scale bar: 1 cm. (F) Cauline leaves from wild-type and atbrm plants (lines 10.1 and 29.1) grown in LD. Scale bar: 1 cm. (G) Inflorescences from wild type and atbrm (line 29.1) plants grown in SDs. Scale bar: 1 mm. (H) Cross section of curled rosette leave from a atbrm plant (line 29.1). Scale bar: 1 mm.

View larger version (151K): [in a new window]   Fig. 5. Flower phenotypes of AtBRM-silenced plants. In all cases plants were grown under SD conditions. (A-D) Scanning electron micrographs of mature wild-type (A) and atbrm (B-D) flowers. Scale bar: 0.5 mm. The mutant flowers have a variety of phenotypes: (B) short petals and stamens, (C) bent carpel, (D) four stamens. The arrowhead in D indicates two fused stamens. (E) Higher magnification of fused stamens shown in D. Scale bar: 250 µm. (F) Gynoecium tip from an atbrm plant, stigma and style are absent. Scale bar: 10 µm. (G,H) Stamen filament cells from mature wild-type (G) and atbrm (H) flowers. Scale bar: 10 µm. (I) Sepaloid petal in an atbrm flower. Scale bar: 0.5 mm. (J) Abaxial cells from the sepaloid petal shown in I. Arrowheads indicate stomatal cells. Scale bar: 10 µm. (K) Abaxial cells from a wild-type petal. Scale bar: 10 µm. The atbrm plants belong to line 10.1 in all cases except C, which is line 29.1.

View larger version (121K): [in a new window]   Fig. 6. Inflorescence meristem in atbrm plants. (A) Wild type (left) and atbrm line 29.1 (right) inflorescences. (B) Wild-type and atbrm line 29.1 inflorescence meristems at days 23, 28, 33 and 38. Stage 1-3 floral buds are numbered from youngest to oldest, in some of the micrographs. Scale bar: 100 µm.

View larger version (88K): [in a new window]   Fig. 7. Early flowering phenotype of atbrm plants (line 29.1). (A) Wild-type and atbrm plants at 40 days growth under SD conditions. (B-E) RNA was isolated from total seedlings, collected 9 hours after dawn, at 10, 13 or 16 days of growth under SD conditions (10 hours light/14 hours dark). RNA blots were hybridized with a FLC- (B) or with a SOC1 (E)-specific probe and reprobed with 18S ribosomal DNA (18S) as a loading control. Levels of CO (C) and FT (D) mRNA were determined by semi-quantitative RT-PCR. Levels of the ubiquitin mRNA were also tested by RT-PCR as control.