Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia

Abstract

We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family.

Figure 1.

Overview of the FS Insertion Screening Strategy. (A) Design of a reverse FS primer (FSP) at the 3′ end of the conserved MADS domain. IRP represents the terminal inverted repeat–based dTph1 transposon primer. (B) Scheme of the three-dimensional pooling strategy for the insertion libraries. Leaves of each plant are harvested three times (e.g., material from plant 1 [P1] is present in the pools consisting of all individuals in the X3 [gray], Y1, and Z3 planes). If P1 contains an insertion in the gene of interest, three fragments amplified with a gene-specific primer and a transposon primer will appear on the gel, one in each dimension and displaying identical sizes. The positioning of these three fragments on the gel yields a unique address (X, Y, and Z coordinates), allowing the identification of a single positive plant in the original population. (C) Alignment of part of the MADS domain of the MADS box family members, for which we recovered insertions using an FS primer (reverse and complement of sequence P122) based on the FBP4 gene. Nucleotide mismatches between primer and templates are shaded gray. (D) Partial gel image from an insertion screening of two insertion libraries with an FS primer based on the FBP4 gene. Amplification products were visualized by labeling the FS primer and sized on a 4.5% polyacrylamide gel. Arrows indicate unique three-dimensional coordinates representing plants that contain insertions in the MADS domain of FBP4, FBP5, FBP9, and PMADS17. M indicates molecular marker lane. (E) Typical direct sequencing results from reamplified fragments. IRP represents the dTph1 transposon primer sequence, and arrows indicate the precise transposon insertion positions.

Figure 2.

Neighbor-Joining Tree of MIKC-Type MADS Box Genes from Petunia, Arabidopsis, Snapdragon, and Selected Members from Other Species. Genes isolated from Arabidopsis, petunia, and snapdragon are shown in black, red, and blue, respectively. All others (Le-RIN, Le-JOINTLESS, and LeTM6 from tomato, ZMM17 from maize, and GGM13 from Gnetum gnemon) are shown in green. Local bootstrap probabilities are indicated for branches with >60% support. The partial sequences of the petunia MADS box genes PMADS6, -10, -16, and -17 were not included in the phylogenetic analysis because this would have resulted in poorly supported trees. However, PMADS6 and -17 cluster with PMADS15, forming a distinct outgroup of the Arabidopsis AGL27-like genes, and PMADS10 and -16 belong to the TM3 subfamily.

Figure 3.

Phenotype of the Petunia sep-Like Mutants fbp2, fbp5, and fbp2 fbp5. (A) Wild-type W138 petunia flower. (B) Flower of fbp2-1 exhibiting a green hue on the corolla. (C) Longitudinal section through an fbp2-1 flower bud of 3 mm showing the emergence of two secondary inflorescences in the third whorl (arrows). a, anther; ov, ovary; p, petal; s, sepal; sec ifl, secondary inflorescence. (D) Close-up of a longitudinal section through an fbp2-1 mutant flower at a later stage of development showing an inflorescence and a secondary flower developing all four floral whorls, positioned near the nectary. a, anther; b, bract; c, carpel; nec, nectary; o, ovule; ov, ovary; p, petal; s, sepal; sec ifl, secondary inflorescence. (E) to (J) Scanning electron microscopy images of wild-type and mutant petunia floral organs. (E) and (H) Wild-type petals showing the characteristic conical petal epidermis cells (adaxial side). (F) and (I) fbp2-1 petals with sepal-like epidermis cells, trichomes, and stomata indicated by arrows (adaxial side). (G) Wild-type sepal with typical sepal epidermis cells, trichomes, and stomata (adaxial side). (J) Close-up of the epidermal surface of leaf-like organs (abaxial side) inside the ovary of an fbp2-2 fbp5-1 flower (see [M]) showing the presence of trichomes and stomata and the conversion of ovule epidermal cells to sepal- or leaf-like epidermal cells. (K) Flowers of fbp5-1, fbp2-2, and fbp2-2 fbp5-1 mutants showing enhanced petal-to-sepal conversion in fbp2-2 fbp5-1. From left to right: fbp5-1, fbp2-2, and fbp2-2 fbp5-1. (L) and (M) Scanning electron microscopy images of the ovaries of fbp2-2 and fbp2-2 fbp5-1 flowers. The carpel wall has been removed to reveal the inner organs. (L) fbp2-2 ovary showing wild-type-appearing ovules. The inset shows a close-up of the ovules. (M) fbp2-2 fbp5-1 ovary displaying leaf-like organs replacing the ovules. (N) to (Q) Comparison of third- and fourth-whorl phenotypes of fbp5-1, fbp2-2, and fbp2-2 fbp5-1. (N) From left to right: fbp5-1, fbp2-2, and fbp2-2 fbp5-1 showing sepaloid anthers and the development of a huge pistil in fbp2-2 fbp5-1 (sepals and petals have been partially removed). (O) Ovaries with removed carpel wall. From left to right: side-view of an fbp2-2 fbp5-1 ovary showing densely packed leaf-like organs, an fbp5-1 ovary with normal ovules, and a longitudinal section of an fbp2-2 fbp5-1 ovary showing the leaf-like structures emerging from the positions normally occupied by ovules. (P) Close-up of anthers showing the partial conversion to sepal-like organs in fbp2-2 fbp5-1 flowers. From left to right: fbp5-1, fbp2-2, and fbp2-2 fbp5-1. (Q) Close-up of the style and stigma of fbp2-2 fbp5-1 showing trichomes and a stigma composed of two unfused carpels. Bars = 500 μm in (C) and (D), 100 μm in (E) to (J), and 1 mm in (L) and (M).