Article Figures & Data
Figures
- Figure 1.
Diagram of the Reporter Constructs Used in the WUS Promoter Analysis.
(A) The putative WUS transcription start site (+1) was determined by RACE PCR. Putative CAAT and TATA boxes and the start codon are underlined.
(B) to (D) For each construct, a scheme is shown at left. At right, the name, the relative staining intensities in the inflorescence meristem (IM), the floral meristem (FM), and the ovule (Ov) [−, none; (+), very faint; +, weak; ++, moderate; +++, strong], and the exact coordinates of the WUS promoter fragments or deletions (Δ) are given. The WUS coding region was replaced by the GUS coding sequence (box).
(B) Diagram of the truncation constructs analyzed.
(C) Diagram of the internal deletion constructs analyzed.
(D) Diagram of the constructs used during the functional definition of cis-regulatory sequences. To generate the reporter constructs, the monomers were multimerized as indicated and fused to -60 CaMV:GUS. D (deletion), G (gain of function), L (little deletion), and S (linker scanning) denote series of constructs.
- Figure 2.
Expression Patterns of WUS:GUS Constructs and Complementation of the Inflorescence Phenotype with Corresponding Genomic Fragments.
Each row shows the expression pattern of the indicated WUS promoter fragment in (from left to right) seedlings, inflorescences, and ovules and the complementation of a homozygous wus-1 mutant plant with the corresponding genomic fragment (right column). Seedlings and inflorescences were stained with GUS with 2 mM Fe-cyanide for 1 d except ScaBst (2 mM, 2 d) and Δ4 (5 mM, 1 d). Ovules were stained with GUS with 5 mM Fe-cyanide for 1 d.
(A) Truncation constructs.
(B) Deletion constructs.
b, floral bud; c, cotyledon; ch, chalaza; f, funiculus; h, hypocotyl; l, leaf; n, nucellus. Arrows indicate the shoot meristem, and the arrowhead indicates the floral meristem. Bars = 0.5 mm (seedling, inflorescence), 30 μm (ovule), 2 mm (genomic constructs, except Δ5), and 5 cm (genomic construct Δ5 and the wild type).
- Figure 3.
Expression Patterns of WUS:GUS Reporter Constructs in Inflorescences.
Inflorescences ([A] to [D] and [F] to [L]) or seedling (E) were stained with either 2 mM Fe-cyanide ([A], [B], and [D] to [H]) or 5 mM Fe-cyanide ([C] and [I] to [L]) in the staining buffer for 1 d ([A] and [H] to [J]) or 3 d ([B] to [G], [K], and [L]).
(A) to (H) Whole-mount views with bright-field optics. GUS activity is visualized by blue color.
(A) Δ51.
(B) (D5)4.
(C) (D5)4 clv1-4.
(D) (G1)4.
(E) (L5)4. Staining in hydathodes (hy) and stipules (st) is attributable to background activity of the included minimal promoter.
(F) (L5)4.
(G) (S0)4.
(H) M31-HindBst (−566/−564 mutated; see Figure 4).
(I) to (L) Eight-micrometer sections viewed with dark-field optics. GUS activity is visualized by pink color.
(I) HindBst.
(J) ScaBst.
(K) (D5)4.
(L) (L5)4.
b, floral bud; fm, floral meristem; im, inflorescence meristem; s, sepal. Arrows indicate the shoot meristem, and arrowheads indicate the floral meristem. Bars = 0.5 mm ([A] and [E]), 2 mm (C), and 60 μm (I). Magnification of (B), (D), and (F) to (H) is as in (A); magnification of (J) to (L) is as in (I).
- Figure 4.
Regulatory Architecture of the WUS Promoter.
The approximate positions of regulatory domains are indicated. At bottom, nucleotides within the 57-bp spatial control region essential for promoter activity in the stem cell niche of the inflorescence meristem (RE1 and RE2) are indicated in boldface letters; the predicted HD-ZIP binding site is underlined. QE, quantitative element required for enhanced expression levels.
- Figure 5.
Linker Scanning Analysis.
Schemes of the constructs analyzed. The 118-bp WUS promoter fragment S0 (−586/−469) was permutated with the decamer sequence ACCTCGAGTC, generating the mutated fragments S1 to S12. The −566/−557 and −546/−537 regions were also scanned with trinucleotide/tetranucleotide exchanges (M31 to M33 and M51 to M53). For the reporter constructs, each mutated fragment was tetramerized and fused to −60 CaMV:GUS. Unaltered nucleotides are indicated with dashes. Relative staining intensities in inflorescence meristems (IM) and floral meristems (FM) are indicated at right for tetrameric (tetramer) and full-length WUS promoter constructs (HindBst). All full-length promoter constructs additionally showed strong staining in ovules unaffected by the indicated mutations.
Tables
- Table 1.
Complementation of wus-1 Mutant Flowers with Promoter Truncations and Internal Deletion Constructs
Whorl Construct Sepals Petals Stamens Carpels n HindBst 4.0 ± 0 4.0 ± 0 5.3 ± 0.4 2.0 ± 0 20 FspBst 4.0 ± 0 4.0 ± 0 5.6 ± 0.5 2.0 ± 0 20 ApaBst 4.0 ± 0 4.0 ± 0 5.8 ± 0.4 2.0 ± 0 20 ScaBst 4.0 ± 0 4.0 ± 0 5.9 ± 0.3 2.0 ± 0 20 Δ1 4.0 ± 0 4.0 ± 0 5.7 ± 0.5 2.0 ± 0 20 Δ2 4.0 ± 0 4.0 ± 0 4.5 ± 1.2 1.3 ± 0.8 20 Δ3 4.0 ± 0 4.0 ± 0 5.9 ± 0.4 2.0 ± 0 20 Δ4 4.0 ± 0 4.0 ± 0 5.9 ± 0.2 2.0 ± 0 18 Δ5 4.5 ± 1.0 4.4 ± 0.8 5.1 ± 1.3 0.1 ± 0.3 20 wus-1a 3.7 ± 0.7 3.6 ± 0.9 0.9 ± 0.5 0.0 ± 0 53 Ler 4.0 ± 0 4.0 ± 0 6.0 ± 0.3 2.0 ± 0 20 The average floral organ number and the sd in n flowers of the line that showed the best complementation are indicated.
↵a Data for wus-1 are taken from Laux et al. (1996).
Supplemental Data
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