Article Figures & Data
Figures
- Figure 1.
The Phenotype of the baf1 Mutant.
(A) Wild-type (wt) tassel.
(B) baf1-ref mutant tassel.
(C) Primary branches from wild-type and baf1-ref tassels. Arrowheads point to single spikelets in the baf1-ref mutant.
(D) Wild-type and baf1-ref mutant ears.
(E) and (F) Internodes subtending mature ears form an indentation in wild-type plants (E) that is absent in baf1-ref mutants (F) (ears have been removed).
(G) In baf1-ref mutants, the ear is borne on a slightly elongated internode (arrowhead). The star marks the prophyll.
(H) Longitudinal section stained with Toluidine Blue of a wild-type seedling, showing the shoot apical meristem (star) and axillary meristems forming at the axils of developing leaves in an alternate arrangement (arrowheads). Bar = 100 μm.
(I) Seedlings of both wild-type and baf1-ref mutant plants were stripped of most leaves, revealing axillary buds (arrowheads) in normal but not in baf1-IL mutant plants.
- Figure 2.
The Fusion Defects in the Development of baf1 Mutant Ears.
(A) Toluidine Blue staining of sections of young axillary meristems during vegetative development in wild-type (wt) and baf1-ref plants. Arrowheads point to the groove between the axillary meristem and the upper leaves. Bar = 50 μm.
(B) In baf1-ref internodes, axillary meristems fused to the culm cause the formation of a notch.
(C) Series of cross sections of developing ears, stained with Safranin O-Alcian Blue, reveal the lack of a separation layer in baf1-ref mutants between the stem and the developing axillary inflorescence. Arrowheads demarcate the separation zone normally observed in wild-type plants. The star marks the prophyll.
Bars = 500 μm.
- Figure 3.
Baf1 Encodes an AT-Hook Transcriptional Regulator.
(A) Schematic representation of the map-based cloning of the Baf1 locus. Boxed are the syntenic regions analyzed for candidate genes in both sorghum and rice.
(B) Amino acid sequence of the BAF1 protein. In green is the amino acid sequence of the AT-hook DNA binding motif, whereas in blue is the PPC domain (DUF296).
(C) Schematic representation of the Baf1 gene and of the lesions identified in the baf1-ref and baf1-IL mutants. The white triangle represents a deletion.
(D) and (E) Transient expression of the BAF1-YFP fusion protein (D) and YFP (E) in tobacco leaves.
- Figure 4.
Phylogenetic Analysis.
Bayesian consensus phylogram of 87 BAF1-like AT-hook DNA binding proteins from phylogenetic analyses comprising 50 million generations and the General Time Reversible model of evolution with invariant sites and gamma distributed rates (GTR+I+G) partitioned according to codon position. Bold branches supported by ≥0.95 posterior probability. Gymnosperms: PINA, Pinaceae; Eudicots: BRAS, Brassicaceae; SALI, Salicaceae; VITA, Vitaceae; Monocots: POAC, Poaceae; ZING, Zingiberaceae.
- Figure 5.
BAF1 Proteins Form Homo- and Heterodimers.
(A) Schematic representation of the constructs used in yeast two-hybrid and in vitro pull-down assays.
(B) Targeted yeast two-hybrid assay. Yeast transformants were grown on selective medium for both bait (BD:BAF1-Δ762) and prey (AD:BAF1, AD:BAF1R-1, and AD) constructs (-LT) and on medium lacking His (-LTH) for detecting interactions. Interactions are also verified by β-galactosidase activity (βgal). AD, GAL4 activation domain; BD, GAL4 DNA binding domain.
(C) and (D) In vitro pull-down assays. BAF1 (C) and BAF1R-1 (D) fused to a 3XHA tag were obtained by in vitro transcription/translation and were incubated with GST-only (negative control) or GST-BAF1 bound beads. Anti-GST antibody shows even loading of GST-only and GST-BAF1 proteins, whereas anti-HA antibody shows preferential retention of the input only on GST-BAF1 beads.
- Figure 6.
The Expression Pattern of Baf1.
(A) to (F) In situ hybridizations of Baf1 during vegetative and reproductive development in the wild type.
(A) Longitudinal section of a seedling showing Baf1 expression adaxial to the newly developing axillary meristems (arrowhead and close-up).
(B) and (C) Localization of Baf1 in an immature tassel (B) and a tassel branch (C) in a narrow domain where the axillary meristems are forming.
(D) A similar localization is observed in immature ears.
(E) and (F) In developing spikelet meristems, Baf1 is localized where a new floral meristem is forming (arrowheads).
(G) to (L) Consecutive sections hybridized with probes for either Baf1 or Zyb15, a marker for suppressed bract primordia.
(G) and (H) flank of an inflorescence meristem. Arrowheads point to a few Baf1-expressing cells in (G) and the corresponding region in (H).
(I) and (J) Immature developing ear.
(K) and (L) Developing spikelet meristems.
Black arrowheads in (I) mark the corresponding P1 and P2 sites of Zyb15 expression in (J). White arrowheads in (K) mark the Baf1 expression domain. P1 and P2, suppressed bract primordia. Bars = 50 μm.
- Figure 7.
The Relationship between Baf1 and Ba1.
(A) and (B) Consecutive sections of an inflorescence meristem of a developing ear hybridized with Baf1 (A) or Ba1 (B) probes show overlapping expression patterns.
(C) and (D) ba1 baf1 double mutant tassels show no noticeable phenotypic differences from single ba1 mutants. Suppressed bracts are still evident in both single and double mutants (close-ups).
(E) In situ localization of Baf1 in a ba1 mutant tassel shows a regular pattern of expression of Baf1.
(F) Quantitative RT-PCR of Ba1 in pools of seedlings of heterozygous or homozygous baf1-IL mutant plants. The expression levels are relative to actin, and the expression in the heterozygous sample is set to 100. Error bars represent sd.
(G) and (H) In situ localization of Ba1 in wild-type (wt) (G) and baf1-IL mutant (H) seedlings. Arrowhead points to Ba1 expression in a developing axillary meristem.
Bars = 50 μm.
- Figure 8.
Model for Baf1 Function in Axillary Meristem Initiation.
In developing inflorescences, the expression of the suppressed bract marker Zyb15 (P1, in blue) is followed by the expression of Baf1 in a boundary domain (P2, in red). Baf1 is required, together with other unknown factors, for proper expression of Ba1. Ba1 triggers axillary meristem initiation at the axils of the suppressed bracts (P2) and the establishment of a population of meristematic cells, expressing Kn1 (in green). IM, inflorescence meristem; AM, axillary meristem. P1, P2, and P3 mark suppressed bract primordia, with P1 marking the youngest primordium.
Tables
Phenotype n Tassel Length (cm) No. of Primary Branches Branch Length (cm) % Single Spikelets on Branches (n > 600) Branch Radial Size (mm; n > 50) Wild type 27 27.94 (2.98) 10.67 (2.47) 15.65 (3.12) 1.9% 1.96 (0.36) baf1-ref 10 23.85 (4.97)* 14.40 (3.47)*** 13.55 (3.85)**** 14.3%** 1.60 (0.46)** Wild type 10 32.25 (1.78) 6.10 (2.13) 16.79 (2.29) 5.1% n.d. baf1-IL 11 28.32 (1.90)** 4.64 (1.03) 11.97 (4.77)** 37%** n.d. Tassel measurements on the wild type and baf1-ref mutants in the B73 background (BC2) and the wild-type and baf1-IL in the A619 background (BC1). Average numbers (sd in parentheses). All compared values are in the same genetic background. *t test, P = 0.041; **t test, P ≤ 0.0001; ***t test, P = 0.0008; ****t test, P = 0.0009. n.d., not determined.
Genotype n No. of Tillers % Earless tb1 27 7.30 (2.41) 0% baf1-IL 20 0.65 (0.81)* 15% tb1 baf1-IL 7 2.14 (1.95)* 42% Quantification of the average number of tillers in tb1 baf1 double mutants (sd in parentheses). All values were compared to the tb1 single mutant. *t test, P < 0.0001.
Clone No. n Homology Gene Model 1 4 AT-hook DNA binding transcriptional regulator GRMZM2G029096 2 1 AT-hook DNA binding transcriptional regulator GRMZM2G024005 3 1 Zinc-finger transcription factor GRMZM2G055116 4 1 Hypothetical protein GRMZM2G097249 List of BAF1Δ762 interactors identified in a yeast two-hybrid library screen.
Supplemental Data
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