The MADS box gene FBP2 is required for SEPALLATA function in petunia

Abstract

The ABC model, which was accepted for almost a decade as a paradigm for flower development in angiosperms, has been subjected recently to a significant modification with the introduction of the new class of E-function genes. This function is required for the proper action of the B- and C-class homeotic proteins and is provided in Arabidopsis by the SEPALLATA1/2/3 MADS box transcription factors. A triple mutant in these partially redundant genes displays homeotic conversion of petals, stamens, and carpels into sepaloid organs and loss of determinacy in the center of the flower. A similar phenotype was obtained by cosuppression of the MADS box gene FBP2 in petunia. Here, we provide evidence that this phenotype is caused by the downregulation of both FBP2 and the paralog FBP5. Functional complementation of the sepallata mutant by FBP2 and our finding that the FBP2 protein forms multimeric complexes with other floral homeotic MADS box proteins indicate that FBP2 represents the same E function as SEP3 in Arabidopsis.

Figure 1.

Arabidopsis and Petunia sep-Like Mutants and Complementation of the Arabidopsis Mutant by FBP2 Overexpression. (A) Wild-type Arabidopsis plant flowering after the production of 9 to 12 rosette leaves (rl). (B) Severe Arabidopsis 35S-FBP2 plant flowering extremely early after the formation of just one curled rosette leaf (rl) and terminating in a single flower (tfl). (C) Flower of the triple sep1 sep2 sep3 mutant, with the formation of sepals in whorls 1, 2, and 3 and a new floral bud developing from the center of the flower (whorl 4). (D) Wild-type petunia (W115) inflorescence. Bracts (b), flowers (f), and inflorescence (i) are indicated. (E) Inflorescence of a petunia fbp2 cosuppression plant. (F) Flower of the petunia fbp2 cosuppression plant, with homeotic conversions identical to those found for the Arabidopsis sep1 sep2 sep3 mutant flower (C). (G) Flower from an Arabidopsis sep1 sep2 sep3 35S-FBP2 plant (No. 270) showing a mild complementation of the sep mutation. Whorl-2 organs are petaloid, and in whorl 4, an aberrant bud appears on a short stem. (H) Flower from another Arabidopsis sep1 sep2 sep3 35S-FBP2 (No. 23) plant, with petals in whorl 2, staminoid organs in whorl 3, and an ovary-like structure in whorl 4 containing stigmatic tissue (st) on top. (I) Late flower from the Arabidopsis sep1 sep2 sep3 35S-FBP2 plant No. 23 with pollen-bearing stamens in whorl 3. Part of the sepals and petals are removed. (J) Close-up of the stamen in (I). (K) Close-up of an ovary-like structure in whorl 4 from a flower of the Arabidopsis sep1 sep2 sep3 35S-FBP2 plant No. 23. The two carpels are fused almost completely and have stigmatic tissue (st) on top. Inside, sepal-like organs develop (not visible). Bars in (A) and (B) = 1 cm.

Figure 2.

Phylogenetic Tree of Selected Members of the AP1 and FBP2/SEP MADS Box Protein Family. The FBP, pMADS, and PFG proteins are from petunia, Le-RIN and TM5 are from tomato, and all others are from Arabidopsis. The phylogenetic analysis was performed with the MADS, I, and K regions of the proteins. Bootstrap values are indicated above each branch. The Arabidopsis AP3 protein (class B) was used as an outgroup.

Figure 3.

Expression Patterns of Petunia FBP2 Family Members in Petunia Wild-Type and FBP2 Cosuppression Plants Determined by RNA Gel Blot Analyses. (A) Expression of FBP2 family members in various tissues of wild-type petunia plants. (B) Expression of FBP2 family members in floral organs of FBP2 cosuppression plants. Both FBP2 and FBP5 are strongly downregulated in these plants. In each lane, 10 μg of total RNA was loaded. Equal loading of RNA was demonstrated by staining a representative gel with radiant red before blotting (rRNA).

Figure 4.

In Situ Localization of FBP2, FBP5, and pMADS12 Transcripts in Wild-Type Petunia. Longitudinal sections were hybridized to digoxigenin-labeled antisense RNA fragments of FBP2 ([A] to [D]), FBP5 ([E] to [H]), and pMADS12 ([I] and [J]). (A) FBP2 expression in a very young floral meristem. No signal is obtained in the inflorescence meristem and bracts. (B) FBP2 expression in the central part of an early-stage floral bud. No signal is present in the developing sepals. (C) Young floral bud with developing sepal, petal, and stamen primordia. The carpel primordia just arise. FBP2 mRNA can be detected in the three inner floral whorls. (D) FBP2 expression in a slightly later stage floral bud than that shown in (C). (E) Same stage meristems as in (A) hybridized to an antisense FBP5 probe. In contrast to FBP2, FBP5 also is expressed in the inflorescence meristem. (F) FBP5 expression in an early floral bud showing a pattern similar to that obtained for FBP2 (B). (G) and (H) FBP5 expression in the inner three floral whorls. Stages are comparable to those shown in (C) and (D), respectively. (I) Expression of pMADS12 in the inflorescence and floral meristem. The expression pattern resembles the expression of FBP5 at this stage (E). (J) pMADS12 expression in a same stage floral bud as that shown in (C) and (G). b, bract; c, carpel; f, floral meristem; i, inflorescence meristem; p, petal; s, sepal; st, stamen. Bars in (A), (B), (E), (F), and (I) = 200 μm; bars in (C), (D), (G), (H), and (J) = 500 μm.