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First published online 21 July 2004
doi: 10.1242/dev.01279

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PETAL LOSS, a trihelix transcription factor gene, regulates perianth architecture in the Arabidopsis flower

Philip B. Brewer, Paul A. Howles^*, Kristen Dorian, Megan E. Griffith, Tetsuya Ishida, Ruth N. Kaplan-Levy, Aydin Kilinc and David R. Smyth

School of Biological Sciences, Monash University, Melbourne, VIC 3800, Australia

View larger version (77K): [in a new window]   Fig. 1. Sepal fusion in mature flowers of single and double mutant combinations of ptl-1, cuc1-1 and cuc2-1. (A) A wild-type (Columbia) flower. (B) A ptl-1 mutant flower with some fusion (<1/4) of adjacent lateral (left) and medial (right) sepals (arrow). Note aberrant shape of sepals (deeper in profile) and the absence of petals. (C,D) Sepal fusions in cuc1-1 (C) and cuc2-1 (D) mutant flowers (arrows). (E,F) Near full fusion of adjacent sepals in ptl-1 cuc1-1 (E) and ptl-1 cuc2-1 (F) double-mutant flowers (arrows). Scale bar: 1 mm.

View larger version (73K): [in a new window]   Fig. 2. cDNA sequence, genomic organization and nuclear localization of PTL. (A) Sequence of PTL cDNA D171 (Accession number AY555728), and the predicted amino acid sequence showing two trihelix DNA-binding domains (shaded), a conserved alpha-helical central domain (dotted line) that includes a predicted coiled-coil domain (solid line), and putative nuclear localization sequences (bold). The sites of the single intron, and of sequence changes in five ptl mutant alleles, are also shown. (The 100 bp deletion in ptl-3 is associated with the insertion of TTTTATGT at the same site.) (B) Genomic organization surrounding PTL, showing putative translated regions (green) and an intron (white). The 10.7 kb clone that fully complemented the ptl mutant phenotype is also shown (intergenic regions – yellow). Five reporter gene constructs carry the GUS gene (blue) translationally fused downstream of PTL genomic regulatory sequences, in some cases including the 1,127 bp intron (white). (C,D) Onion epidermal cells showing nuclear localization of the PTL protein translationally fused downstream of GFP (C), compared with location of control GFP protein alone (D). Left, bright field; right, GFP fluorescence of same field. Scale bars in C,D: 100 µm.

View larger version (50K): [in a new window]   Fig. 3. Expression patterns of PTL assessed using RT-PCR. All tissues were from the Columbia ecotype except lane 11 (Ler). The same total RNA samples were used for three reactions in each case, using mRNA specific primers for either PTL (top), flower-specific APETALA3 amplified using essentially the same conditions (centre), or ACTIN2 as a control (bottom). 3.5 µl of the reaction was loaded for PTL and AP3 reactions, 1.5 µl for the ACT2 control. In lane 6, the receptacle at the base of the siliques was removed before RNA extraction.

View larger version (124K): [in a new window]   Fig. 4. Expression patterns of PTL using in situ hybridization and GUS reporter genes. (A) Location of DIG-labelled antisense PTL RNA (brown) hybridized in situ with PTL mRNA in transverse section of an inflorescence meristem. Label is concentrated in inter-sepal zones (arrows) and sepal margins. (The black deposit centred between the two arrows is a staining artefact.) M, shoot apical meristem; 4, 5 and 6, bud stages. (B-N) Location of GUS reporter gene product in wholemounts (blue) or in sections (pink, dark field). (B) Side view of young inflorescences showing four spots of staining in young flower primordia (p8.0i::GUS). (C) Transverse section of inflorescence showing absence of early staining in the flower primordium without the intron, although sepal margin expression still occurs (p8.0::GUS). (D-H). Five serial transverse sections of an inflorescence showing staining patterns in buds from stage 1 to 6 (indicated in D). Staining is also present in the edges of cauline leaves (cl) (p2.0i::GUS). (I,J). Petal primordia (p) are not stained at stage 6 (I) or 7 (J). ls, lateral stamen (p2.0i::GUS). (K) Longitudinal section of stage 9 bud showing staining in the basal margins of a developing petal (p2.0i::GUS). (L) Wholemount of a stamen dissected from a stage 8 flower, showing lateral staining where developing anther and filament adjoin (arrows) (p2.0i::GUS). (M) Young seedling viewed from above showing staining in edges of developing leaves, initially all round, but later limited to basal regions (p2.0i::GUS). (N) Transverse section of shoot apical meristem of young seedling showing expression in leaf margins (arrows) and stipules (asterisks), but none in the shoot apical meristem (M) (p2.0i::GUS). Scale bars: A,C,L,N, 100 µm; B,M, 500 µm; in D, 500 µm for D-H; I-K, 50 µm.

View larger version (150K): [in a new window]   Fig. 5. Consequences of overexpression and ectopic expression of PTL. (A) ptl-1 mutant inflorescence. (B) Complementation of ptl-1 mutant phenotype in pPTL(1.3i)>>PTL plant (moderate driver line #5). (C) Gain-of-function phenotype in pPTL(1.3i)>>PTL ptl-1 plant (strong driver line #7). Lateral sepals are absent, and four petals are restored, either separate (SEM inset), or fused along their lateral margins. (D,E) SEMs of inflorescence apex of (D) wild-type plant and (E) gain-of-function pPTL(1.3i)>>PTL ptl-1 plant (strong driver line #1). Buds at stages 4, 5 and 6 are indicated. Lateral sepals are either absent or narrow in gain-of-function plants (E, arrows). (F) SEM of pAP1>>PTL plant with inflorescences producing flowers arrested at stage 2. (G) pAP3>>PTL flower showing absence of petals and stamens, and reduced lateral sepal. (H) SEM of pAP3>>PTL flower showing absence of petal and stamen primordia in the second and third whorls. ca, carpel; f, filamentous structure; se, sepal. (I) p35S>>PTL seedling showing final extent of growth. Strongly inducible PTL target line #10 was used for all ectopic expression plants (F-I). Scale bars: A-C,G, 1 mm; inset in C, D-F,H,I, 100 µm.

View larger version (99K): [in a new window]   Fig. 6. Expression of PTL in pinoid mutant plants. (A) pid-2 mutant inflorescence. (B) p2.0i::GUS expression in pid-2 inflorescence. Rings of staining (blue) occur in the first whorl region of two buds (arrows). (C) Transverse section of a developing pid-2 flower at the same stage as that shown in B. M, shoot apical meristem. (D) Longitudinal section of an older pid-2 flower showing GUS staining (pink) accumulating in the inner region of the expanding first whorl (arrows). Scale bars: A, 1 mm; B-D, 100 µm.

View larger version (22K): [in a new window]   Fig. 7. Summary of expression patterns of PTL (shaded) in lateral regions of flower primordia (stages 1-4, vertical view), and in developing leaves and floral organs (lateral view).

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