Control of corolla monosymmetry in the Brassicaceae Iberis amara

Abstract

Establishment of morphological novelties has contributed to the enormous diversification of floral architecture. One such novelty, flower monosymmetry, is assumed to have evolved several times independently during angiosperm evolution. To date, analysis of monosymmetry regulation has focused on species from taxa where monosymmetry prevails, such as the Lamiales and Fabaceae. In Antirrhinum majus, formation of a monosymmetric corolla is specified by the activity of the TCP transcription factors CYCLOIDEA (CYC) and DICHOTOMA (DICH). It was shown that establishment of monosymmetry likely requires an early asymmetric floral expression of CYC homologs that needs to be maintained until late floral stages. To understand how CYC homologs might have been recruited during evolution to establish monosymmetry, we characterized the likely CYC ortholog IaTCP1 from Iberis amara (Brassicaceae). Species of the genus Iberis form a monosymmetric corolla, whereas the Brassicaceae are otherwise dominated by genera developing a polysymmetric corolla. Instead of four equally sized petals, I. amara produces two small adaxial and two large abaxial petals. The timing of IaTCP1 expression differs from that of its Arabidopsis homolog TCP1 and other CYC homologs. IaTCP1 lacks an asymmetric early expression but displays a very strong differential expression in the corolla at later floral stages, when the strongest unequal petal growth occurs. Analysis of occasionally occurring peloric Iberis flower variants and comparative functional studies of TCP homologs in Arabidopsis demonstrate the importance of an altered temporal IaTCP1 expression within the Brassicaceae to govern the formation of a monosymmetric corolla.

Fig. 1.

Development of the monosymmetric corolla in I. amara. Different stages of Iberis flower development are shown. (A–C) Developmental stages before anthesis were monitored under the SEM; sepals were removed to reveal inner organs. (A) After simultaneous initiation, adaxial (aD) and abaxial (aB) petal (p) primordia are of equal size. Central stamen primordia arise slightly before the lateral stamen primorida, developing between the adaxial and abaxial petals. The gynoecium appears as a central dome. (B) At the onset of stamen differentiation, when filament and anther become distinguishable, adaxial and abaxial petals start to differ in size, with the abaxial petals being slightly larger. (C) Petal size difference slowly increases until anthesis of the flower. A lateral stamen is removed in B and C. (D) After flower opening, the petal size difference strongly increases from the young stage A1 to the fully mature stage A2 (E). (F) The young inflorescence of Iberis shows a corymboid architecture that gives it the appearance of one single, large flower. (Scale bars: A–C, 50 μm; D and E, 5 mm.)

Fig. 2.

RT-PCR analysis of IaTCP1 expression in vegetative and floral organs. (Upper) The graph depicts average IaTCP1 transcript values from three independent reactions, normalized to the expression strength of IaRan3. Error bars indicate standard deviations. IaTCP1 is weakly expressed in leaves (lf) and shoots (st), as well as in inflorescences (inf), flowers before anthesis (fl bA), mature flowers (fl A2), and gynoecia (gyn). A dynamic differential expression becomes apparent in separately analyzed adaxial (aD) and abaxial (aB) petals isolated from flowers before anthesis (bA), from young flowers after anthesis (A1), and from fully mature flowers (A2). Expression is always stronger in adaxial than in abaxial petals. (Lower) Gel image of a representative IaTCP1 and IaRan3 RT-PCR.

Fig. 3.

In situ expression pattern of IaTCP1 and IaH4 in young Iberis flowers. (A–C) IaTCP1 antisense probe hybridized to a longitudinal section through the Iberis inflorescence meristem and young developing flowers. (A) No distinct asymmetric IaTCP1 expression is detectable in the inflorescence meristem and adjacent young floral primordia (fp). (B) After onset of stamen differentiation, weak IaTCP1 expression becomes visible in petals (p) where similar expression levels were detected in adaxial (aD) and abaxial (aB) petals. Expression was also observed in anthers (lateral anther, la). (C) An asymmetric IaTCP1 expression becomes apparent in later floral stages, where a stronger signal was detectable in adaxial (arrow) than in abaxial petals. (D) Antisense IaH4 probe was hybridized to serial sections shown in C revealing that more cells express IaH4 in abaxial petals (arrowhead) compared with adaxial ones, which indicates higher cell proliferation activity in abaxial petals. (Scale bars: 200 μm.)

Fig. 4.

A peloric flower variant of I. amara develops an abaxialized corolla. (A) Wild-type flower at stage A2. (B) Naturally occurring peloric flower variant with only abaxialized petals. (C) Representative RT-PCR result showing reduced IaTCP1 expression in peloric (pl) compared with wild-type (wt) corollae. Quantification of four RT-PCRs revealed that >27-fold (27.64 ± 9.81) more IaTCP1 transcript was detectable in wild-type petals compared with peloric petals. For normalization, IaRan3 expression level was determined.

Fig. 5.

Constitutive expression of IaTCP1, TCP1, and CYC in Arabidopsis affects petal growth. Front view of flower and habitus of wild type (A) and representative transgenic plants expressing IaTCP1 under the control of the CaMV35S promoter (B and C). (B) Transgenic plants with an intermediate phenotype form smaller petals and show enhanced secondary shoot outgrowth. (C) Plants with a strong phenotype often develop small flowers lacking mature floral organs and show a dwarfed vegetative growth. (D) Transgenic 35S::TCP1 plants with an intermediate phenotype produce smaller petals and resemble flowers overexpressing IaTCP1 (B). (E) Contrarily, constitutive CYC expression increases petal sizes in transgenic plants. (F) Gel image of an RT-PCR shows that IaTCP1 expression strength correlates with phenotype strength. For RT-PCR analysis, inflorescences were harvested from wild-type (wt) plants as well as intermediate (im) and strong (st) phenotype plants, segregating in a T2 population derived from selfing the intermediate phenotype T1 plant 35S::IaTCP1/2. For normalization, expression of IaRan3 was analyzed. (Scale bars: 2 mm.)