Article Figures & Data
Figures
- Fig. 1.
Alignment of the predicted amino acid sequences for LtMADS1 and LtMADS2 with some other members of the AP1/SQUA branch: Arabidopsis, AP1(accession no. Z16421) and FUL (accession no. U33473);Oryza sativa, OsMADS14 (accession no.AF058697) and OsMADS15 (accession no. AF058698); andZea mays, ZAP1 (accession no. L46400). Dashes indicate gaps to maximize alignment. Amino acids that are identical in four or more of the proteins are blocked. The MADS box is amino acids 1 through 60, the I region is amino acids 61 through 93, and the K box is amino acids 94 through 170.
- Fig. 2.
Phylogenetic tree based on complete MADS box domain sequences of selected members of the plant MADS box gene family. Apart from including more recent sequences the analysis and subfamily designations were based on Theissen et al. (1996). Sequences: Arabidopsis (AP1, CAL, FUL,AGL3, AG, and AGL20); snapdragon (SQUA and DEFH24); barley (Hordeum vulgare; BM3, BM5, andBM8); Brassica oleracea (BoAP1);Dendrobium globulus (DoMADS1, DoMADS2, and DoMADS3); Eucalyptus sp.(EAP1 and EAP2); L. temulentum(LtMADS1 and LtMADS2); maize (Zea mays; ZAG1, ZAP1, ZMM4, and ZMM5); potato (Solanum tuberosum;POTM1); rice (OsMADS1, OsMADS14 [also known asRAP1B], OsMADS15 [possibly the same asRAP1A], and OsMADS45); Silene latifolia (SLM4 and SLM5); Sinapis alba (SaMADSA, SaMADSB, andSaMADSC); sorghum (Sorghum bicolor; SbMADS1 andSbMADS2); tobacco (Nicotiana tabacum; TobMADS1), tomato (Lycopersicum esculentum; TM3, TM4, andTM8), and wheat (Triticum aestivum; TaMADS11; accession no. AB007504).
- Fig. 3.
Scheme of alternative splicing events used to generate LtMADS2 and LtMADS2′ transcripts. A, The boxed sequence indicates the open reading frame (ORF) with stop codons indicated with an X and UTR as lines. Changes in the amino acid sequence of the putative translated protein from theLtMADS2′ transcript compared with the LtMADS2transcript is indicated by the black (intron) and stippled boxes (frameshifted original ORF). Spliced introns are shown as triangles. B, Nucleotide sequence at the intron/exon 4 junction. Intron sequence was determined by genomic sequencing. Putative splice sites forLtMADS2 and LtMADS2′ transcripts are indicated with arrows. b, Bases.
- Fig. 4.
Gel-blot analysis of LtMADS1 andLtMADS2 expression in leaves, roots, and shoot apices ofL. temulentum using the same gene specific probes forLtMADS1 (A) and LtMADS2 (B) as for in situ hybridization. Total RNA (20 μg) from leaves or roots was blotted and hybridized (A and B), then both blots were stripped and reprobed (C) and with a 9-kb wheat 26S rRNA clone, pTA71 (D; Gerlach and Bedbrook, 1979). For analysis of shoot apex mRNAs, PCR-based cDNA product (virtual northern) from mRNA of short day (SD; vegetative) or LD III (florally induced) apices was hybridized with the gene specific probe for LtMADS1 (E) or LtMADS2 (F). Sizes in kb are indicated between the blots.
- Fig. 5.
A, Floral score, assessed by dissection, ofL. temulentum shoot apices as a function of time after a single inductive LD (Day II). Values are means ±se, n = 10; some error bars are hidden by the symbols. Vertical arrows are positioned to indicate developmental stages. A three-dimensional pattern of expression ofLtMADS1 mRNA (B) obtained by in situ hybridization was generated by apex reconstructions obtained by stacking/combining the images from four to five longitudinal sections up to the median one for a vegetative (SD) or LD IV early double-ridge stage apex (single median images shown in Fig. 6, B and C, respectively). Images were digitized and cropped using Adobe Photoshop and stacked using Confocal Assistant 4.02. The bar is 200 μm.
- Fig. 6.
Expression of LtMADS1 andLtMADS2 RNA in L. temulentum shoot apices, assessed by in situ hybridization. All sections were hybridized with the antisense riboprobe except for A and K, which show representative sections hybridized with a sense control riboprobe. All sections were hybridized and processed together; therefore, the intensity of antisense probe staining reflects the expression of the genes. Apex stages: vegetative shoot apices (SD; A, B, I, and J); early double ridge (LD IV; C, K, and L); lateral spikelet sites on the flanks of the shoot apex at glume/lemma stage (LD XII; D and M) or the floret stage (LD XXI; E); terminal spikelet site subtended by two glume primordia with three floret meristems positioned alternately along the terminal spikelet axis subtended by lemma primordia (LD XXX; F and O); three lateral spikelet sites at floret stage (LD XXX; G and N); and a lemma, anterior stamen, and carpel primordium (left to right) on the flank of a floret site (LD XXX; H and P). The bars are 50 μm. am, Apical meristem; as, anterior stamen primordium; c, carpel; f, floret; g, glume; l, lemma; lp, leaf primordium; pr, provascular strand; sm, spikelet meristem; tsm, terminal spikelet meristem.
- Fig. 7.
Expression of LtLFY RNA in L. temulentum shoot apices assessed by in situ hybridization. Sections A through C, E, and F were hybridized with the antisense riboprobe. A representative section hybridized with the sense control riboprobe is shown in D. Magnified images of B are shown in E and F. The bars are 50 μm. Apex stages and other conditions as for Figure6.
- Fig. 8.
Scanning electron micrographs of flowers from plants expressing LtMADS1, LtMADS2, and LtMADS2′under the control of a 1.7-kb fragment of the AP1 promoter. A, Flower from LtMADS1 transgenic plant with two petaloid bracts in the perianth (arrows). The inset in A shows petaloid cells on the margin of the lower bract-like petal at high magnification (size bar = 100 μm). B, Flower from LtMADS2 transgenic plant. A first-whorl bract has been removed to reveal a second-whorl bract (arrow). C, Inflorescence from LtMADS2′ transgenic plant. An axillary flower with an abnormally long pedicel is visible in the upper right hand corner. D, Enlargement of flower in lower left hand corner of C. Size bars are 1 mm, except 200 μm in C.
Tables
- Table I.
Phenotype of ap1-15 mutants expressing LtMADS1, LtMADS2, or LtMADS2′ under the control of the AP1 promoter
Transgene None LtMADS1 LtMADS2 LtMADS2′ Whorl 1 Total organs 2.2 ± 0.3 2.5 ± 0.9 4.5 ± 0.3 2.8 ± 0.3 Sepals 0.0 0.2 ± 0.8 0.0 0.0 Bracts 2.0 ± 0.2 2.2 ± 1.0 4.3 ± 0.2 2.8 ± 0.3 Filamentous organs 0.1 ± 0.1 0.1 ± 0.3 0.2 ± 0.3 0.1 ± 0.1 Whorl 2 Total organs 1.0 ± 0.3 1.2 ± 1.1 2.6 ± 0.6 1.0 ± 0.2 Petals 0.0 0.0 0.0 0.01-a Petaloid bracts 1.0 ± 0.3 1.2 ± 1.1 1.3 ± 0.6 1.0 ± 0.2 Leaves 0.0 0.0 1.3 ± 0.7 0.0 Whorl 3 Total organs 5.1 ± 0.3 5.2 ± 1.1 6.6 ± 0.5 5.7 ± 0.2 Stamens 4.1 ± 0.4 4.8 ± 1.1 5.7 ± 0.8 4.8 ± 0.3 Mosaic stamens 0.9 ± 0.4 0.3 ± 0.7 1.0 ± 0.7 0.9 ± 0.3 Filamentous organs 0.1 ± 0.1 0.2 ± 0.5 0.1 ± 0.2 0.1 ± 0.1 Flower No. 28 86 22 84 2° Flowers/inflorescences 1.6 ± 0.3 1.6 ± 0.1 0.3 ± 0.3 1.8 ± 0.1 Averages with 2 × se of the mean are given. Increased organ no. in whorls 1 and 2 and reduced no. of secondary flowers/inflorescences in LtMADS2, when compared with the other three genotypes, are statistically significant (Student'st test, p < 0.001).
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↵F1-a One petal in 84 flowers.
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