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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00620

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Phyllotactic pattern and stem cell fate are determined by the Arabidopsis homeobox gene BELLRINGER

Mary E. Byrne^1,*, Andrew T. Groover^2,*, Joseph R. Fontana² and Robert A. Martienssen^1,

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
² Institute of Forest Genetics, Davis, California 95616, USA

View larger version (53K): [in a new window]   Fig. 1. blr mutant phenotype. (A) Wild-type Landsberg erecta and (B) blr-1 mutant plants. blr has additional leaves and flowers, is reduced in stature and has precocious outgrowth of axillary meristems. Phyllotaxy is also disturbed. (C) In the wild-type inflorescence siliques are arranged in a regular spiral phyllotactic pattern. (D) In blr mutant inflorescences siliques occur in aberrant positions and internodes are irregular.

View larger version (114K): [in a new window]   Fig. 2. Phyllotaxy defect in blr mutants. (A,C,E,G) Wild-type inflorescence apex; (B,D,F,H) blr mutant inflorescence apex. (A,B) Longitudinal sections of 23-day-old plants showing comparable size and shape of wild-type (A) and blr (B) inflorescence meristems. The width of the stem is also similar in wild type and blr. (C-F) 18-day-old plants. In wild type (C,E) organs are initiated on average 137.5° apart forming a continuous spiral. In wild type (C) the divergence angle between primordia 2 and 3 is 130.6°. In blr mutants (D,F) flowers can initiate in aberrant positions as in primordia 3 in D and 5 in F. In D the divergence angle between primordia 2 and 3 is 79.3°. (G,H) Inflorescence apex from 23-day-old plants. Clockwise (red) and counterclockwise (yellow) contact parastichies connecting floral primordia in a 3+5 phyllotactic pattern. Additional organs in blr mutants result in contact parastichies forming a tighter curve than in wild type. Numbering of each inflorescence starts at the youngest visible primordium. Scale bars: 0.5 mm (A,B), 50 µm (C,D), 200 µm (E,F) and 500 µm (G,H).

View larger version (40K): [in a new window]   Fig. 3. blr mutant alleles. (A) Diagram of the BLR gene is with locations of Ds insertions in blr-1 and blr-2, and the T-DNA insertion in blr-3 indicated. (B) RT-PCR amplification using gene-specific primers and hybridization of products with gene-specific probes. BLR transcripts are detected in wild type but not in the three mutants (top panel). RBC transcripts were amplified as a control (bottom panel). (C) Northern hybridization detects low levels of BLR transcript in wild-type seedling and leaf tissue and much higher levels in the inflorescence and flowers. No wild-type transcript is detected in the blr-1 mutant. Lower panel shows an ethidium bromide-stained gel reflecting relative amounts of RNA.

View larger version (75K): [in a new window]   Fig. 4. BLR expression pattern. (A,B) Whole mounts showing GUS expression pattern from the BLR::GUS reporter gene. GUS activity is detected in the SAM of the embryo (A) and 8-day-old seedling (B). Weak expression is also in the vasculature of cotyledons and leaves. (C,D) In situ hybridization of wild-type embryos probed with BLR antisense (C), and STM antisense (D). BLR and STM transcripts are detected in the SAM (E,F) In situ hybridization of wild-type inflorescences probed with BLR antisense (E) and STM antisense (F). (G-I) Three consecutive serial sections of an inflorescence apex probed with BLR antisense. BLR is expressed in the SAM, in a pattern similar to that of STM. In initiating floral primordia BLR is first downregulated (arrowheads in G-I) but is subsequently detected in the central region of developing floral primordia and flowers (arrows in E). A low level of BLR expression is also detected at the base of developing flowers, in the pedicel and in the inflorescence stem. Scale bars: 50 µm (A,C-I).

View larger version (94K): [in a new window]   Fig. 5. blr enhances a weak allele of stm. Twelve-day-old seedlings of stm-11 (A), blr stm-11 (B), stm-2 (C) and blr stm-2 (D). blr stm-11 double mutants are similar to stm-11 single mutants. No vegetative shoot is produced and the base of the cotyledons are fused. Plants homozygous for the weak stm-2 allele show limited fusion at the base of the cotyledons and initiate a vegetative shoot. The meristem defect is enhanced in blr stm-2 double mutants which do not develop vegetative shoot structures and there is significant fusion of the cotyledons.

View larger version (51K): [in a new window]   Fig. 6. blr interacts with as stm1 but not with bel1. (A,B,F,G,H) Whole plants and (C-E) seedlings of bp (A), blr bp (B), blr as1 (C), blr as1 stm-11 (D-F), bel1 (G) blr bel1 (H). (A) The bp mutant is reduced in stature, pedicels are short and flowers hang down. (B) The blr bp double mutant is much reduced in stature compared with either single mutant. Double mutants also have additional leaves, pedicels are short and siliques tend to hang down (inset in B). (C) Twelve-day-old emerging shoot of blr as1 seedling compared with (D) blr as1 stm-11 sibling, with only a single vegetative leaf. (E,F) Occasionally vegetative shoot growth is resumed in blr as1 stm-11 triple mutants. Leaves in the double and triple mutant have an as1 phenotype. (G) The phenotype of the bel1 mutant is confined to the ovule. Siliques do not elongate as plants fail to set seed. (H) The double blr bel1 mutant fails to set seed as in bel1 single mutants. The phyllotaxy is disturbed and stature is reduced as in blr single mutants. Scale bars: 2.5 cm (A,B,F-H).

View larger version (33K): [in a new window]   Fig. 7. BLR interacts directly with class 1 KNOX proteins. Yeast two-hybrid assay demonstrating interaction between BLR and KNOX proteins inferred through selective growth on medium lacking leucine, tryptophan and histidine (-leu -;trp -;his) compared with medium lacking leucine and tryptophan (-;leu, -;trp). All yeast strains grow on -;leu -;trp medium. Growth on -;leu, -;trp, -;his medium is detected for strains carrying TA-BLR and DB-BP or DB-STM but not for strains carrying TABLR and DB-KNAT4 or the DB vector. Growth of a strain carrying TA-BEL1 and DB-BP is shown as a positive control.