Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage

Abstract

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.

Figure 1.

Phenotypes Observed in Various P. hybrida B-Class and bl Mutant Combinations. (A) to (D) Top views of wild-type W138 (A) and Petunia def (gp) (B) flowers and closeup top views of bl (C) and def bl (D) flowers. Arrows in (C) and (D) indicate second whorl antheroids and second whorl antheroids terminating with a short style/stigma structure, respectively. (E) to (H) Side views of wild-type W138 (E), def (gp) (F), bl (G), and def bl (H) flowers. Some sepals were removed in (E), (F), and (H) to reveal the inner organization. Note that the front sepal in (G) has a carpelloid tip. (I) and (J) Top view (I) and detail (J) of a Petunia tm6/+ def flower showing proliferating anther tissue in the third whorl terminating in a short style/stigma structure (arrow). (K) Petunia tm6 def double mutant flower showing full conversion of stamens to carpels, forming a central congenitally fused chimney-like structure. (L) Flower of a Petunia TM6-RNAi line in a def mutant background showing a similar phenotype as in tm6 def flowers. (M) and (N) Top view (M) and detail (N) of tm6/+ def bl flowers showing proliferating anther tissue in the third whorl and carpelloid structures in the second whorl (arrow in [N]). Sepals were removed in (N) to reveal inner organs. (O) A tm6 def bl triple mutant flower showing full conversion of petals and stamens to carpelloids, forming a central multiwhorled, congenitally fused chimney-like structure. (P) Flower of a TM6-RNAi line in a def bl mutant background showing a similar phenotype as in tm6 def bl flowers.

Figure 2.

Expression Analysis of P. hybrida B- and C-Class MADS Box Genes in Floral Whorls of Wild-Type and Various Mutant Flowers as Determined by Real-Time PCR. Organ types are indicated above bars showing gene expression levels as follows: se, sepal; pe, petal; st, stamen; ca, carpel. Homeotically converted organs are shown underlined. All reactions were performed in duplicate using a biological replicate for each sample (represented by pairs of gray and black bars). The height of the bars for a given gene indicates relative differences in expression levels within the range of tissues tested. For each primer combination separately, the highest expression value encountered in the series of different tissues tested was set equal to 100, and lower values are plotted relative to this highest value on a linear y axis scale. GAPDH (for glyceraldehyde-3-phosphate dehydrogenase) expression levels were used for normalization.

Figure 3.

Petunia TM6 Overexpression Phenotypes in Wild-Type and def Mutant Backgrounds. (A) 35S:TM6 overexpression in wild-type Petunia showing the development of ectopic petals on the outer surface of the petal tube. (B) Detail of first whorl organs in 35S:TM6 plants showing a partial conversion to a petal tube-like structure. (C) 35S:TM6 overexpression in a def mutant background showing partial and more complete complementation of petal development. The arrow indicates the main petal veins retaining sepal identity in a strongly complemented overexpression line. (D) to (F) Scanning electron microscopy images of the adaxial epidermis of def second whorl sepalloids showing the typical sepal epidermal characteristics, such as jigsaw-shaped epidermal cells interspersed by trichomes and stomata (D), wild-type petals showing the characteristic conical petal cells (E), and second whorl petals of def mutants complemented with 35S:TM6 (F). Bars = 100 μm.

Figure 4.

Alignment of Conserved Regions Identified in euAP3- and TM6-Type 5′ Putative Regulatory Sequences. Species names are abbreviated as follows: Ph, Petunia hybrida; Sl, Solanum lycopersicum; Mo, Misopates orontium; Am, Antirrhinum majus; At, Arabidopsis thaliana; Cp, Cochlearia pyrenaica. Accession numbers of these sequences can be found in Methods. Numbers flanking the fragment names indicate their positions relative to the ATG start codon: (1), position of the three proposed CArG boxes in the Arabidopsis AP3 promoter (Hill et al., 1998; Tilly et al., 1998); (2), position of the proposed CArG box in the Antirrhinum DEF promoter; (3), mutation site of the chlorantha def allele (Schwarz-Sommer et al., 1992).

Figure 5.

Neighbor-Joining Tree of Newly Isolated euAP3 and TM6 Homologs from Various Solanaceous Species, Including a Selection of B-Class Lineage MADS Box Genes from Other Informative Taxa. Members of the GLO/PI subfamily were used as an outgroup, and 1000 bootstrap samples were generated to assess support for the inferred relationships. Local bootstrap probabilities of >70% are shown near the major branching points. euAP3 and TM6 putative proteins from solanaceous species included in this analysis are indicated with the extension SOL-x, of which the x corresponds to the lettering on the images of the flowers of these species at left.

Figure 6.

Model of B-Function Regulation in P. hybrida.