Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development

Abstract

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.

Figure 1.

Simplified angiosperm phylogeny paired with the gene phylogeny of AP1/FUL homologs and their C-terminal motifs. This figure is based on Litt and Irish (2003) and N. Pabón-Mora and A. Litt (unpublished data). A, Simplified angiosperm phylogeny showing the five major groups of flowering plants (basal angiosperms, monocots, basal eudicots, rosids, and asterids) and indicating the phylogenetic positions of Papaver and Eschscholzia. The phylogenetic positions of Arabidopsis, Antirrhinum (snapdragon), and Triticum (wheat) are also shown. B, Simplified AP1/FUL gene lineage tree, showing the core eudicot gene duplication. The bottom dotted box indicates the groups in which FUL-like genes are found, which includes all taxa outside of the core eudicots. The top dotted box shows the euAP1 and euFUL genes in core eudicots. Arabidopsis AP1/FUL homologs (AP1, CAL, FUL, AGL79) are shown in the gene tree, as are PapsFL1, PapsFL2, EscaFL1, and EscaFL2. In the center are the C-terminal protein motifs typical of the gene groups depicted in B: the euAP1 transcriptional activation (Trans-Act) and farnesylation (Farn) motifs and the FUL-like motif characteristic of euFUL and FUL-like proteins.

Figure 2.

Expression of PapsFUL-like genes at different developmental stages. A, RT-PCR results showing the expression of PapsFL1 and PapsFL2 in dissected floral organs at different floral bud stages and in flowers at anthesis as well as in fruits and leaves. Stages are based on Drea et al. (2007). At bud stage P7, petal primordia have not elongated; at bud stage P8, petals are fully expanded inside the floral bud. B to O, In situ mRNA hybridization. B to F, Expression of PapsFL1. G to K, Expression of PapsFL2. L to O, Expression common to PapsFL1 and PapsFL2 (L, PapsFL1 section; M–O, PapsFL2 sections). B and H, Early floral meristem; sepal primordia are starting to differentiate. C and I, Floral bud with large sepals protecting the incipient petal, stamen, and carpel primordia. D and J, Floral bud with overlapping sepals and fully differentiated petal, stamen, and carpel primordia. E, Floral bud with clearly defined anther and filament. F and K, Longitudinal section of the carpel in a preanthesis floral bud. G, Shoot apical meristem before the transition to flowering. L, Cross-section of the young fruit showing the fruit wall. M and N, Longitudinal section of the ovary showing placenta and ovules. O, Dormant axillary bud subtended by the lowermost cauline leaves. c, Carpel; cl, cauline leaf; fr, fruit; ii, inner integument; l, leaf primordia; lf, leaf; n, nucellus; p, petal; s, sepal; st, stamen. Arrows indicate petal primordia, and asterisks indicate carpel primordia. Bars = 50 μm (B–E, H–J, and O), 70 μm (G), 150 μm (F and K ), 100 μm (L), and 120 μm (M and N).

Figure 3.

Expression of EscaFUL-like genes at different developmental stages. A, RT-PCR results showing the expression of EscaFL1 and EscaFL2 in floral buds (P7 and P8) and different parts of the flower at anthesis as well as in leaves and fruits. B to M, In situ mRNA hybridization of EscaFL1 and EscaFL2. Expression of EscaFL1 and EscaFL2 is identical (B, E–H, and J–M, EcFL1 sections; C, D, G, I, and N, EscaFL2 sections). B, Inflorescence meristem protected by two highly dissected cauline leaves. C and D, Axillary inflorescence showing the terminal flower before sepal initiation (C) and at the beginning of sepal initiation (D). E, Axillary inflorescence showing a terminal flower with sepal primordia and a floral meristem axillary to the cauline leaf on the left. F, Floral bud with elongating sepals enclosing petal, stamen, and carpel primordia. G, Floral bud with fused sepals around petal, stamen, and carpel primordia. H, Floral bud with fully closed carpel wall. I, Floral bud with sporogenous tissue in the stamens. Sepals were forced open artificially in this sample. J, Tips of the highly dissected mature cauline leaves. K to M, Early stages of ovule development showing the nucellus (K), the initiation of the two integuments (L), and the fully formed bitegmic ovules (M). c, Carpel; cl, cauline leaf; f, axillary floral meristem; fr, fruit; ii, inner integument; im, inflorescence meristem; lf, leaf; n, nucellus; p, petal; s, sepal; st, stamen. Arrows indicate petal primordia, and asterisks indicate carpel primordia. Bars = 60 μm (B–F), 100 μm (G–J), and 50 μm (K–M).

Figure 4.

Inflorescence and cauline leaf phenotypes in opium poppy plants treated with TRV2-PapsFL1 and TRV2-PapsFL2 separately. A, Wild-type opium poppy. B, Plants down-regulated for papsfl1. C, Plants down-regulated for papsfl2. D to F, Leaf clearings of cauline leaves in wild-type opium poppy (D), papsfl1 plants (E), and papsfl2 plants (F). Two cauline leaves are presented in each panel to illustrate the variation of size and shape from larger, more basal cauline leaves (left) to smaller, more apical cauline leaves (right). White arrows indicate axillary branches. Bars = 5 cm (A–C) and 1 cm (D–F).

Figure 5.

Floral phenotypes in opium poppy plants treated with TRV2-PapsFL1 and TRV2-PapsFL2 separately. A, Wild-type opium poppy flower after deciduous sepals have fallen. B and C, Flowers showing persistent leaf-like sepals that remain attached to the base of the flower in papsfl1 plants (B) and papsfl2 plants (C). D, Wild-type sepals. E and F, SEM of the abaxial (E) and adaxial (F) wild-type sepal surfaces. G, Persistent papsfl1 leaf-like sepals during fruit development. H and I, SEM of the abaxial (H) and adaxial (I) leaf-like papsfl1 sepal surface. J, Persistent papsfl2 leaf-like sepals during fruit development. K and L, SEM of the abaxial (K) and adaxial (L) leaf-like papsfl2 sepal surface. M, Wild-type opium poppy petals. N and O, SEM of the abaxial (N) and adaxial (O) wild-type petal surfaces. P, papsfl1 flower showing small green patches on the abaxial surface of the outer petals. Q and R, SEM of the petaloid abaxial (Q) and adaxial (R) papsfl1 petal surfaces. S, papsfl2 flowers showing small green areas in the distal portion of the petal. T and U, SEM of the petaloid abaxial (T) and adaxial (U) papsfl2 petal surfaces. V, Wild-type immature opium poppy fruit (poricidal capsule formed by eight fused carpels). W, Cross-section of the wild-type fruit showing main vascular bundles of each placenta. X, papsfl1 fruit. Y, Cross-section of papsfl1 fruit. Z, papsfl2 fruit. AA, Cross-section of papsfl2 fruit. p, Placenta. Black arrows point to vascular traces in the pericarp, and white arrows point to green patches on the petals. Bars = 1.5 cm (A–C), 0.5 cm (D, G, J, P, V, X, and Z), 40 μm (E, F, H, I, K, L, N, O, Q, R, T, and U), 0.7 cm (M and S), and 500 μm (W, Y, and AA ).

Figure 6.

Floral phenotypes in opium poppy plants treated with TRV2-PapsFL1 and TRV2-PapsFL2 simultaneously. A to C, Double down-regulated plants showing large green areas in the two outer petals in opium poppy. A, Front view. B and C, Side views. D, Dissected papsfl1-papsfl2 green petal. E and F, SEM of the abaxial (E) and adaxial (F) papsfl1-papsfl2 petal surface. G, Dissected wild-type petal. H and I, SEM of the abaxial (H) and adaxial (I) wild-type petal surface. J, Dissected wild-type carpel. K and L, SEM of the abaxial (K) and adaxial (L) surface of young wild-type carpel. M, Dissected wild-type leaf. N and O, SEM of the abaxial (N) and adaxial (O) surface of wild-type leaf. Bars = 0.75 cm (A–C), 0.5 cm (D, G, J, and M), 50 μm (E, F, H, I, N, and O), and 30 μm (K and L).

Figure 7.

Floral phenotypes in California poppy plants treated with TRV2-EscaFL1 and TRV2- EscaFL2. A to C, Wild-type California poppy plant (A) and flower showing the persistent floral cup after sepal abscission (B) and fruit (C). D to F, Down-regulated escafl1-fl2 plant showing increased branching compared with the wild type (D) and leaf-like organs replacing the sepals and persisting after anthesis (E) and during fruit development (F). G, Wild-type cauline leaf. H and I, SEM of the abaxial (H) and adaxial (I) wild-type cauline leaf surface. J, Cauline leaves in escafl1-fl2 plants. K and L, SEM of the abaxial (K) and adaxial (L) escafl1-fl2 cauline leaf surface. M, Wild-type fused sepals before anthesis. N and O, SEM of the abaxial (N) and adaxial (O) wild-type sepal surface. P, Homeotic transformation of sepals into leaf-like organs. Q and R, SEM of the abaxial (Q) and adaxial (R) leaf-like escafl1-fl2 sepal surface. S, Dehisced wild-type fruit. T, Cross-section of the wild-type fruit before dehiscence showing the ring of lignified tissue and the two dehiscence zones. U, escafl1-fl2 fruit showing premature rupture of the fruit wall and exposure of the immature seeds. V, Cross-section of the escafl1-fl2 fruit showing the lignified ring interrupted by a thinner, weaker section of the pericarp through which the fruit ruptures. White arrows point to the abscission zone between the floral cup and the deciduous portion of the sepals; arrowheads point to the dehiscence zones in the fruit. e, Endocarp; ect, ectopic lignification; sd, seed. Bars = 3 cm (A and C), 0.5 cm (B, D–F, I, R, and T), 0.3 cm (L and O), 20 μm (G, H, J, K, M, N, P, and Q), and 0.1 mm (S and U).

Figure 8.

Optimization and mapping of functions recorded for AP1/FUL homologs. Based on the available data, we hypothesize that the ancestral functions in the AP1/FUL gene lineage include floral meristem and sepal identity (1), because these are functions that are shared with the sister SEPALLATA and AGL6 gene lineages. Transition to the reproductive meristem (2) appears to be ancestral just to the AP1/FUL lineage. Before the diversification of the Papaveraceae, the genes acquired functions in cauline leaf development (3), branching (6), and fruit development (5), although the acquisition of these functions could have happened earlier than is shown here. After the diversification of the core eudicots, some of these functions (1 and 6) were retained by both the euFUL and the euAP1 clades, whereas others (2, 3, and 5) were exclusively retained by members of the euFUL clade. A role in petal identity (4) appears to have been independently acquired in opium poppy and Arabidopsis. Asterisks indicate that no functional data are available. FMI/SEP, Floral meristem identity and sepal identity; T REP/F TIME, transition to reproductive meristems/flowering time; C LEAF, cauline leaf development; PET, petal identity; FR, fruit development; BR, branching. Black circles indicate gain of function (also numbered on the right), and white circles indicate loss of function. + symbolizes that the function has been recorded for that gene.