Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia

Abstract

Angiosperms display a wide variety of inflorescence architectures differing in the positions where flowers or branches arise. The expression of floral meristem identity (FMI) genes determines when and where flowers are formed. In Arabidopsis thaliana, this is regulated via transcription of LEAFY (LFY), which encodes a transcription factor that promotes FMI. We found that this is regulated in petunia (Petunia hybrida) via transcription of a distinct gene, DOUBLE TOP (DOT), a homolog of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Mutation of DOT or its tomato (Solanum lycopersicum) homolog ANANTHA abolishes FMI. Ubiquitous expression of DOT or UFO in petunia causes very early flowering and transforms the inflorescence into a solitary flower and leaves into petals. Ectopic expression of DOT or UFO together with LFY or its homolog ABERRANT LEAF AND FLOWER (ALF) in petunia seedlings activates genes required for identity or outgrowth of organ primordia. DOT interacts physically with ALF, suggesting that it activates ALF by a posttranslational mechanism. Our findings suggest a wider role than previously thought for DOT and UFO in the patterning of flowers and indicate that the different roles of LFY and UFO homologs in the spatiotemporal control of floral identity in distinct species result from their divergent expression patterns.

Figure 1.

Analysis of dot Mutants. (A) Wild-type inflorescence. (B) Scanning electron micrograph of a wild-type inflorescence apex. Note that flowers at two developmental stages are seen. The youngest (bottom left) still lacks visible organ primordia, while the oldest (top) has generated primordia for sepals, petals (not visible), and stamens. (C) dot inflorescence. Note the proliferation of bracts and the absence of flowers. (D) Scanning electron micrograph of a dot inflorescence apex. (E) Map of DOT and mutant alleles. Yellow triangles indicate dTPH1 insertions; the green triangle indicates a dTPH7 insertion. All insertion alleles exhibit identical phenotypes. (F) and (G) Inflorescence (F) and flower (G) of the weak dot^H2082/A2232 mutant. ap, apex; ax, axillary meristem; br, bract; f1 and f2, flowers 1 and 2; pe, petal; se, sepal; st, stamen. Bars = 100 μm.

Figure 2.

Molecular Analysis of Tomato an-1 and an-3 Mutants. (A) Inflorescence from wild-type tomato. (B) Inflorescence from an-1 tomato. Note that no flowers are formed but instead the inflorescence only generates new IMs, leading to a cauliflower-like structure. (C) RT-PCR analysis of mRNAs from wild-type (+) and an (−/−) tomato inflorescences. (D) PCR analysis of DNA from two homozygous wild-type (+) and two an-1 (−) tomato plants, showing the genomic disruption in the an-1 allele. PCR used primers complementary to FA (primer pair A) or distinct regions of Sl DOT (primer pairs B to H; shown in [E]). (E) Map of Sl DOT/Sp DOT showing the position of the mutations in an-1 and an-3. Solid lines indicate fragments that can be amplified from an-1 with the indicated primer pair; dotted lines denote fragments that cannot be amplified from an-1 (see Supplemental Table 1 online for primer sequences). (F) PCR analysis using primer pair I (see [E]) of a homozygous wild-type plant, the an-3 mutant, and a heterozygous plant cosegregating for the an phenotype (+ for the wild type and − for the mutant).

Figure 3.

In Situ Localization of DOT mRNA in Petunia. (A) to (D) Expression of DOT mRNA in a vegetative apex (A) and inflorescence apices and young FMs of different stages ([B] to [D]). (E) ALF expression in the section adjacent to that in (D). (F) and (G) Expression of DOT (F) and ALF (G) in adjacent sections through a young FM. (H) DOT expression in a late FM. DOT expression ceases shortly after. (I) and (J) DOT expression in a dot^A2232 (I) and an alf (J) inflorescence. (K) ALF expression in a dot inflorescence. br, bract; ca, carpel; ct, cotyledon; “FM,” FM that is homeotically transformed into an IM; lp, leaf primordium; pe, petal; se, sepal; st, stamen. Bars = 100 μm.

Figure 4.

Phenotype of 35S:DOT Petunia Transformants. (A) Wild-type and 35S:DOT plants making their first flowers. The wild-type plant is about twice as old as 35S:DOT (45 versus 25 d, respectively). (B) The 35S:DOT inflorescence is a solitary flower. (C) and (D) Scanning electron micrographs of a 35S:DOT inflorescence apex. Note that the lateral IM is missing and that the flower contains supernumerary petals and stamen primordia. Sepals, petals, and stamens are indicated by green, red, and yellow asterisks, respectively. (E) and (F) Carpel–stamen chimeras formed in whorl 4 of some 35S:DOT flowers. In (E), anthers develop within the ovary. The carpel in (F) is almost completely converted into a stamen with some ovules along the filament. (G) Sepals of a 35S:DOT transformant in a background that specifies red coloration of petal limbs. (H) Leaves of wild-type and 35S:DOT plants in a background specifying pale red coloration of petal limbs. (I) Scanning electron micrograph of a 35S:DOT leaf, showing a region with leaf epidermal cells and trichomes (right side of image) and a region with epidermal petal cells and lacking trichomes (left side of image; cf. [J] and [K]). (J) and (K) Epidermal cells from a wild-type petal limb (J) and leaf (K). (L) Flower from the wild type (bottom) and a 35S:DOT Arabidopsis ecotype Columbia line. ca, carpel; ov, ovules; se, sepal. Bars = 100 μm.

Figure 5.

DOT and UFO Interact with ALF and LFY. (A) Activation of a GAL4-responsive LacZ reporter gene in yeast strain expression fusions of GAL4^BD to ALF and LFY or fragments thereof. Numbering indicates amino acid residues. (B) and (C) Activation of a GAL4-responsive LacZ gene in yeast strains expressing different GAL4^BD and GAL4^AD fusions. (D) Confocal images of subepidermal petal cells after coinfection with constructs expressing fusions of DOT, ALF, and PSK1 and the N-terminal or C-terminal part of YFP (YFP^N or YFP^C) or, as a negative control, YFP^N and YFP^C alone. The arrows mark strongly fluorescent nuclei. Bars = 10 μm.

Figure 6.

Genetic Interaction of ALF and DOT. (A) Comparison of wild-type, 35S:DOT, and 35S:DOT alf phenotypes. The arrows indicate the stage when the plants switched from vegetative to reproductive growth. (B) Phenotypes of wild-type (WT) and growth-arrested 35S:DOT 35S:ALF and 35S:UFO 35S:LFY petunia seedlings. ct, cotyledon; l, leaf; lp, leaf primordium.

Figure 7.

Regulation of Organ Identity Genes by DOT. (A) RT-PCR analysis of RNA extracted from entire seedlings with different genotypes (see Supplemental Table 2 online for primer sequences). (B) In situ hybridization of the Ph GLO mRNA in the apex of a 35S:ALF 35S:DOT seedling. (C) In situ hybridization of FBP14 mRNA in the apex of a 35S:ALF 35S:DOT seedling. (D) to (F) Double label in situ hybridization of DOT mRNA ([D] to [F]) and mRNA from the B gene Ph GLO (D), the C gene FBP14 (E), and the E gene FBP5 (F). DOT mRNA is seen as a red signal, and RNAs from Ph GLO, FBP14, and FBP5 are seen as brown signal. The inset diagrams depict top views of the plane and position (black line) of the section on the inflorescence, as deduced from examination of a complete series of sections. The red circles indicate the FM and older flowers (f1), and the blue circle represents the IM. br, bract; ca, carpel; pe, petal; se, sepal; st, stamen. Bars = 100 μm.

Figure 8.

Model Explaining the Role of DOT in the Onset of Flowering and the Patterning of Flowers. (A) Model for the role of DOT in the activation of distinct classes of organ identity genes. The white blocks at the top indicate organ primordia in whorls 1 to 4 (w1 to w4). Expression patterns of various mRNAs and proteins are indicated by shaded bars. Black color denotes a high concentration, and lighter (gray) color indicates a lower concentrations. The distribution patterns of DOT/UFO and TER/WUS proteins are hypothetical, as indicated by the question marks. A-type organ identity genes similar to AP1 have not been identified in petunia, as indicated by the question marks. (B) Model explaining the disparate phenotypes of homologous petunia and Arabidopsis mutants. The graphs depict gene expression (vertical axis) during plant development (horizontal axis). The arrows indicate the onset of flowering. ALF expression in the absence of DOT results in inactive protein, as indicated by the unshaded portions.