Flower Development and Perianth Identity Candidate Genes in the Basal Angiosperm Aristolochia fimbriata (Piperales: Aristolochiaceae)

Abstract

Aristolochia fimbriata (Aristolochiaceae: Piperales) exhibits highly synorganized flowers with a single convoluted structure forming a petaloid perianth that surrounds the gynostemium, putatively formed by the congenital fusion between stamens and the upper portion of the carpels. Here we present the flower development and morphology of A. fimbriata, together with the expression of the key regulatory genes that participate in flower development, particularly those likely controlling perianth identity. A. fimbriata is a member of the magnoliids, and thus gene expression detected for all ABCE MADS-box genes in this taxon, can also help to elucidate patterns of gene expression prior the independent duplications of these genes in eudicots and monocots. Using both floral development and anatomy in combination with the isolation of MADS-box gene homologs, gene phylogenetic analyses and expression studies (both by reverse transcription PCR and in situ hybridization), we present hypotheses on floral organ identity genes involved in the formation of this bizarre flower. We found that most MADS-box genes were expressed in vegetative and reproductive tissues with the exception of AfimSEP2, AfimAGL6, and AfimSTK transcripts that are only found in flowers and capsules but are not detected in leaves. Two genes show ubiquitous expression; AfimFUL that is found in all floral organs at all developmental stages as well as in leaves and capsules, and AfimAG that has low expression in leaves and is found in all floral organs at all stages with a considerable reduction of expression in the limb of anthetic flowers. Our results indicate that expression of AfimFUL is indicative of pleiotropic roles and not of a perianth identity specific function. On the other hand, expression of B-class genes, AfimAP3 and AfimPI, suggests their conserved role in stamen identity and corroborates that the perianth is sepal and not petal-derived. Our data also postulates an AGL6 ortholog as a candidate gene for sepal identity in the Aristolochiaceae and provides testable hypothesis for a modified ABCE model in synorganized magnoliid flowers.

Keywords: AGAMOUS-like6; APETALA3; Aristolochia fimbriata; FRUITFULL; MADS-box genes; PISTILLATA; magnoliids; perianth.

FIGURE 1

Aristolochia fimbriata. Anthetic flower in face (A) and back (B) view. Close-up of the gynostemium in female (C) and male (D) phases. Black arrowheads indicate anthers; asterisks (^∗) indicate the upper portion of a single carpel alternating with each anther; l, limb; o, ovary; t, tube; u, utricle. Scale bar: 1 cm.

FIGURE 2

Aristolochia fimbriata, perianth development. (A,B) Shoot apex with floral stages S1 to S4. (C,D) Initiation of sepal primordia (stage S1). (E,F) Elongation of sepal primordia (S2). (G) Perianth closure and ovary (ov) differentiation (S3). (H) Initiation of hooked trichomes (S4). (I) Differentiation of utricle (u), tube (t), and limb (lb) during S5. (J,K) Perianth closure (S6). Black arrows indicate the median sepal; white arrows indicate lateral sepals; arrowheads indicate the shoot apical meristem; ab, accessory bud. Scale bars: (A,B), (G–K): 100 μm; (C,D): 20 μm; (E,F): 40 μm.

FIGURE 3

Aristolochia fimbriata, gynostemium and ovary development. (A,B) Stamen initiation above the forming inferior ovary at S3. (C,D) Anther growth and formation of the sporogenous tissue at S4. (E–G) Two successive stages of gynostemium development at S5 (E) and S6 (F,G). (H) Transverse section of the gynostemium at S6. (I–K) Top (I) and lateral (J,K) views of the “female” stage of gynostemium with stigmatic lobes growing above the staminal tissue at S7. (L) Transverse section of the gynostemium at S7 with carpellary tissue growing between the anthers. (M–O) Successive stages of ovary development at S6 (M,N) and S7 (O). (P) Ovary during transition to fruit (P). o, ovule; ov, ovary; st, stamen; u, utricle; black arrowheads indicate anthers; asterisks (^∗) indicate stigmas. Scale Bars (A,B,L): 60 μm; (C,E–G,J,O,P): 100 μm; (D,H,M): 50 μm; (I), 200 μm; (K), 300 μm; (N), 80 μm.

FIGURE 4

Aristolochia fimbriata, epidermal differentiation of the perianth. (A) Preanthethic flower, longitudinal section. (B–E) Fimbriae development and ornamentation. (F–I) Ornamentation of the abaxial (F) and adaxial (G–I) epidermis of the limb. (J–M) Ornamentation of the abaxial (J) and adaxial (K–M) epidermis of the tube. (N–Q) Ornamentation of the abaxial (N) and adaxial (O–Q) epidermis of the utricle. Scale bars: (B,E): 200 μm; (C,D,O): 100 μm; (F–N,P): 20 μm; (P): 50 μm.

FIGURE 5

Maximum likelihood tree of MADS-box genes identified in the transcriptome of A. fimbriata (arrows). Branch colors denote gene clades, as follows: blue, SEPALLATA; green, AGAMOUS-Like 6; purple, APETALA1/FRUITFULL; ochre, SEEDSTICK; orange, AGAMOUS/SHATTERPROOF; pink, PISTILLATA; red, APETALA3. BS values above 50% are placed at nodes. Asterisks indicate bootstrap (BS) of 100. Besides A. fimbriata, ingroup sequences include those of Amborella trichopoda (Amborellaceae), Saruma henryi (Aristolochiaceae), Aquilegia coerulea (Ranunculaceae), Mimulus guttatus (Phrymaceae), and Arabidopsis thaliana (Brassicaceae), representing the ANA grade, the magnoliids, the basal eudicots, the asterids and the rosids, respectively.

FIGURE 6

Expression profiles of AfimFUL, AfimSEP1, AfimSEP2, AfimAGL6, AfimAP3, AfimPI, AfimAG, and AfimSTK at five different flower developmental stages (A–E), leaves (lf) and capsules (c). ACTIN (AfimACT) was used as a positive control. Measurements below each stage correspond to the total length of the perianth. From S7 onward (B), flowers were dissected into gynostemium (g), limb (l), ovary (o), tube (t), and utricle (u).

FIGURE 7

In situ hybridization of AfimFUL. (A) Flowering shoot apex; note broad expression in floral stages S1 through S4. (B) Floral primordium. (C,D) Flower buds during perianth initiation and early elongation at S1–S2. (E,F) Stamen initiation at S3 (E) and elongation at S4 (F); note broad expression in perianth and gynostemium. (G,H) Gynostemium and ovary differentiation at S5 (G) and S7 (H); note late expression (in H) restricted to perianth, anthers, and ovary. Black arrows indicate medial sepal; black arrowheads indicate anthers; white arrowhead indicates shoot apical meristem (SAM); asterisks indicate stigmas; ab, accessory bud; l, leaf; ov, ovary; o, ovules. Scale bars: (A–C): 100 μm; (D–H): 200 μm.

FIGURE 8

In situ hybridization of AfimAGL6. (A,B) Flowering shoot apex in longitudinal (A) and cross (B) sections; note onset of expression in the perianth. (C–E) Successive stages S3–S6 showing stamen initiation at S3 (C), stamen elongation at S4 (D), and ovary development with ovule differentiation at S7 (E); note expression restricted to perianth and ovary. (F,G) Cross- sections of a floral bud at the limb (F) and the utricle/gynostemium (G) levels; note expression restricted to the perianth. (H) Longitudinal section of the ovary. (I,J) Longitudinal (I) and cross (J) sections of the ovules; note expression in the ovary wall and the integuments. Black arrows indicate medial sepal; black arrowheads indicate anthers; white arrowhead indicates shoot apical meristem; asterisks indicate stigmas; ab, accessory bud; l, leaf; o, ovules; ov, ovary. Scale bars: (A,B,E): 100 μm; (C,D,F,G): 50 μm; (H–J): 60 μm.

FIGURE 9

In situ hybridization of AfimAP3. (A,B) Flowering shoot apex in longitudinal sections; note expression restricted to stamens. (C–E) Successive stages S5-S7 showing initiation of the stigmatic lobes at S5 (C), and elongation at S6–S7 (D,E); note expression restricted to stamens. Black arrows indicate medial sepal; black arrowheads indicate anthers; asterisk (^∗) indicates a stigma; l, leaf; lb, limb; ov, ovary; t, tube; u, utricle. Scale bars: (A–D): 100 μm; (E): 150 μm.

FIGURE 10

In situ hybridization of AfimPI. (A) Flowering shoot apex in longitudinal section; note expression in stamens and surrounding perianth tissue. (B) Stamen initiation at S3. (C) Stamen growth at S4. (D,E) Utricle, tube and limb differentiation together with stigmatic tissue initiation at S5; note expression in the young gynostemium. (F–J) Longitudinal sections and details of a floral bud at the utricle/gynostemium level at S7 (H) and the distal-most adaxial portion of the utricle (I); note expression in the adaxial epidermis, trichomes and hypodermis. Black arrows indicate medial sepal; black arrowheads indicate anthers; white arrowhead indicates shoot apical meristem (SAM); asterisks indicate stigmas; ab, accessory bud; l, leaf; o, ovules; ov, ovary. Scale bars: (A–D,F,G): 100 μm; (E): 150 μm; (H,I,J): 50 μm.