Evolutionary Co-Option of Floral Meristem Identity Genes for Patterning of the Flower-Like Asteraceae Inflorescence

Abstract

The evolutionary success of Asteraceae, the largest family of flowering plants, has been attributed to the unique inflorescence architecture of the family, which superficially resembles an individual flower. Here, we show that Asteraceae inflorescences (flower heads, or capitula) resemble solitary flowers not only morphologically but also at the molecular level. By conducting functional analyses for orthologs of the flower meristem identity genes LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) in Gerbera hybrida, we show that GhUFO is the master regulator of flower meristem identity, while GhLFY has evolved a novel, homeotic function during the evolution of head-like inflorescences. Resembling LFY expression in a single flower meristem, uniform expression of GhLFY in the inflorescence meristem defines the capitulum as a determinate structure that can assume floral fate upon ectopic GhUFO expression. We also show that GhLFY uniquely regulates the ontogeny of outer, expanded ray flowers but not inner, compact disc flowers, indicating that the distinction of different flower types in Asteraceae is connected with their independent evolutionary origins from separate branching systems.

Figure 1.

Expression domains of GhLFY and GhUFO in the IM and FM of wild-type gerbera. A, Expression of GhLFY is absent from the vegetative shoot apical meristem (SAM) and leaves. B, GhLFY shows uniform expression in the young, naked, dome-shaped IM after reproductive transition. C, GhLFY expression marks the incipient flower primordia before their outgrowth (arrow). D, GhLFY first localizes to the adaxial side of incipient bract primordia and later to the axil (arrows) of the elongated involucral bract (iB). E, Expression of GhUFO is lacking from the IM. F, GhUFO expression correlates with FM initiation but occurs later than GhLFY expression (C). G to K, Expression domain of GhLFY during early floral developmental stages. M to Q, Expression domain of GhUFO during early floral developmental stages. L and R, Negative controls were hybridized with sense probes for GhLFY (L) and GhUFO (R). ad, Adaxial; AS, antisense; Ca, carpel; Pa, pappus (sepals of individual flowers); Pe, petal; SE, sense; St, stamen. Bars = 50 µm.

Figure 2.

Phenotypes and expression analysis of wild-type gerbera and transgenic lines with suppressed FMI gene expression. A, Mature inflorescence of nontransgenic gerbera. B, Nontransgenic gerbera from the abaxial side. The inflorescence is surrounded by green involucral bracts. C, Phenotype of a strong transgenic GhLFY RNAi line. D, Phenotype of a mild transgenic GhLFY RNAi line. E, Phenotype of a strong transgenic GhUFO RNAi line. F, Phenotype of a mild transgenic GhUFO RNAi line. G, Expression analysis of three independent transgenic lines with suppressed GhLFY expression compared with wild-type (WT) gerbera. H, Expression analysis of two independent transgenic lines with suppressed GhUFO expression compared with wild-type gerbera. Bars = 1 cm (A–F).

Figure 3.

IM phenotypes in transgenic lines with suppressed FMI gene expression. A, Wild-type IM. The center of the expanding IM has not yet been consumed by emerging flower primordia. B, The IM in the GhLFY RNAi line shows random initiation of flower primordia. C, The IM in the GhUFO RNAi line develops similar to that in the wild type (A). D, Later stage of wild-type IM. The IM is fully consumed with disc flower primordia. E, The IM in the GhLFY RNAi line is never consumed with flower primordia. F, Later stage of IM in the GhUFO RNAi line. The IM is similarly consumed with flower primordia as in the wild type (D). Bars = 500 μm.

Figure 4.

Constitutive expression of GhUFO confers a floral fate to the capitulum. A, General phenotype of the transgenic 35S:GhUFO inflorescence. B, The young capitulum is elongating in 35S:GhUFO rather than expanding as in the wild type. C, Ectopic GhUFO leads to highly modified floral structures with organ primordia emerging in a whorled phyllotaxis. In this line with a strong phenotype, instead of single flowers, floral organs are initiated from the margins toward the center of the capitulum. D and E, A mild phenotype (D) and closeup of the mild phenotype (E) showing that normal flower primordia (FP) are first initiated at the capitulum periphery. The fused ring-like meristem produces petals (Pe) surrounded by pappus bristles (Pa). The innermost whorls are occupied by staminode-like organs (Std) and a mixture of stamen-like structures and staminoid carpels (St/Ca). F, Epidermal cell structures of floral organs in wild-type (WT) and 35S:GhUFO plants. In 35S:GhUFO, the petaloid involucral bracts (iB) show mixtures of petal- and bract-like cell types; the fused ring-like meristem shows petal identity: the sterile staminodes are as in wild-type ray flowers, and functional stamens are as in wild-type disc flowers. We also found variants showing staminoid carpels in the center. G, Expression of GGLO1 (B function MADS box gene) and GAGA2 (C function MADS box gene) in wild-type FM and 35S:GhUFO meristem. B gene expression is confined to petaloid bracts and petals, while both B and C genes are expressed in the staminode/stamen-like organs (St). Bars = 1 cm (A and B), 100 µm (C–F), and 50 µm (G).

Figure 5.

Early ontogeny of ray primordia initiation in wild-type and transgenic gerbera with suppressed FMI gene functions. A to C, Three consecutive developmental stages of early capitulum development in wild-type gerbera. Trans flowers initiate earlier than ray primordia (arrow) that emerge in the axils of the last series of involucral bracts (green asterisks). D to F, SEM images show that the ray flower initials (shaded in yellow) of GhLFY RNAi plants (E) are distinct from the solitary ray primordia (shaded in red) found in wild-type (D) and GhUFO RNAi (F) plants. G to I, In contrast to wild-type (G) and GhUFO RNAi (I) plants, the marginal ray flower primordia in GhLFY RNAi (H) plants show faster organogenesis compared with nearby trans flower primordia. Bars = 50 µm (A–C) and 500 µm (D–I).

Figure 6.

Patterning of the individual flower primordia in transgenic GhLFY and GhUFO RNAi lines. A and B, Transgenic GhLFY RNAi (A) and GhUFO RNAi (B) plants with severe phenotypes form primary primordia (P1; shaded in yellow) that repeatedly initiate secondary (P2) and tertiary (P3) primordia (shaded in orange) in all flower types (ray, trans, and disc). C and D, Patterning of flower primordia in GhLFY RNAi (C) and GhUFO RNAi (D) transgenic plants with milder phenotypes shows flower type-specific responses. In both lines, the ray flower primordia uniformly initiate secondary primordia and, consequently, secondary flowers. The response in trans and disc flowers shows opposite effects: in GhLFY RNAi lines, the disc primordia, and in GhUFO RNAi lines, the trans primordia, pattern as normal flowers (shaded in red). Bars = 100 µm. E, Heat map of quantitative RT-PCR results shows expression profiles of the B, C, and E function MADS box genes in developing primary (R1, T1, and D1) and secondary (R2, T2, and D2) primordia in different flower types. D, Disc flower primordia; M, mild phenotype; R, ray flower primordia; S, severe phenotype; T, trans flower primordia; WT, wild type.

Figure 7.

Phenotypes and expression analysis of the gerbera mutant cv Pingpong. A to C, The inflorescence of cv Pingpong shows similarity to the transgenic GhLFY and GhUFO RNAi lines (A). Extensive proliferation of the inflorescences (B and C) causes splitting of the head. D, Patterning of the capitulum of cv Pingpong. The marginal primordia develop as in wild-type gerbera. E, The inflorescence of cv Pingpong is fully consumed by emerging flower primordia at later developmental stages. F to H, Patterning of the single primordium in cv Pingpong. The single primordium (F) produces bract-like structures surrounding the secondary primordia (G) that further initiate tertiary primordia (H). Bars = 1 cm (A–C) and 100 µm (D–H). I, Relative expression levels of GhUFO in young inflorescences of the wild type (WT) and cv Pingpong (PP). J, Relative expression levels of GhLFY in young inflorescences of the wild type and cv Pingpong. K to M, Relative expression levels of B class (K), C class (L), and SEP-like (M) MADS box genes in primary (P-1) and secondary (P-2) primordia dissected from cv Pingpong in comparison with the wild-type stage 3 flower primordia. Error bars indicate sd calculated from three biological replicates.

Figure 8.

Functional diversification of LFY during capitulum development. A, In Arabidopsis racemes, TFL1 activity regulates the indeterminacy of the IM while LFY defines FMI. In petunia cymes, the IM terminates in a flower. FMI is defined by DOT, and the growth of the inflorescence continues from a sympodial IM (SIM) defined by EVG activity. In gerbera, GhLFY expression is uniform in the determinate IM that subdivides into single flower primordia, where FMI is defined by GhUFO. B, Suggested evolutionary pathway for capitulum development. Species representing Calyceraceae, a close relative of Asteraceae, typically show branched cymose units (marked with asterisks) in the periphery of their inflorescences (Pozner et al., 2012). LFY has evolved a specific role to suppress branching in the marginal ray flower primordia of Asteraceae, as evidenced by the GhLFY RNAi lines.