On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development

Abstract

The ABC model of flower development explains how three classes of homeotic genes confer identity to the four types of floral organs. In Arabidopsis thaliana, APETALA2 (AP2) and AGAMOUS (AG) represent A- and C-class genes that act in an antagonistic fashion to specify perianth and reproductive organs, respectively. An apparent paradox was the finding that AP2 mRNA is supposedly uniformly distributed throughout young floral primordia. Although miR172 has a role in preventing AP2 protein accumulation, miR172 was reported to disappear from the periphery only several days after AG activation in the center of the flower. Here, we resolve the enigmatic behavior of AP2 and its negative regulator miR172 through careful expression analyses. We find that AP2 mRNA accumulates predominantly in the outer floral whorls, as expected for an A-class homeotic gene. Its pattern overlaps only transiently with that of miR172, which we find to be restricted to the center of young floral primordia from early stages on. MiR172 also accumulates in the shoot meristem upon floral induction, compatible with its known role in regulating AP2-related genes with a role in flowering. Furthermore, we show that AP2 can cause striking organ proliferation defects that are not limited to the center of the floral meristem, where its antagonist AG is required for terminating stem cell proliferation. Moreover, AP2 never expands uniformly into the center of ag mutant flowers, while miR172 is largely unaffected by loss of AG activity. We present a model in which the decision whether stamens or petals develop is based on the balance between AP2 and AG activities, rather than the two being mutually exclusive.

Fig. 1.

Expression of miR172. (A-G) Col-0 wild type. (A) Apices from plants grown in short days and transferred to long days to induce flowering. Days after shift are indicated at the bottom. (B) Inflorescence meristem (asterisk) with flanking stage 1 and 2 floral primordia. (C) Stage 4 flower. (D) Stage 5 flower. (E) Stage 7 flower. (F) Stage 8 flower. (G) Developing ovules with signal in integuments. (H-L) ag-2. (H) Inflorescence meristem (asterisk) with flanking stage 1 and 2 floral primordia. (I) Stage 5 flower. (J) Stage 6 flower. (K) Approximately stage 7 flower. (L) Later stage flower. (M) dcl1-11 inflorescence apex (asterisk). se, sepal; pe, petal; st, stamen; gy, gynoecium; int, integuments. Scale bars: 50 μm.

Fig. 2.

Expression of AP2. (A-H) Col-0 wild type. (A) Inflorescence meristem (asterisk), with flanking stage 1 and 2 floral primordia. (B) Stage 3 flower. (C) Late stage 3 flower. (D) Stage 4 flower. (E) Stage 5 flower. (F) Stage 6 flower. (G) Stage 9 flower. Expression of AP2 is present in petals, stamen filaments and placenta with developing ovules. (H) Inflorescence apex hybridized with sense probe. (I,J) Ler-1 wild-type inflorescence apex (I) and cross section through an approximately stage 12 flower (J). (K) Longitudinal section of vegetative Ler-1 apex. (L) Transverse section. AP2 expression is found in emerging leaf primordia on the flanks of the shoot apical meristem (asterisk). In developing leaves, AP2 expression is strongest laterally and adaxially. (M-P) Transgenic plants carrying a pAP2:AP2::YFP reporter. Entire inflorescence (M), cross-section through an approximately stage 12 flower (N), and higher magnification of stage 4 (O) and 5 (P) flowers. There is strong YFP signal (yellow) in the sepals from stage 4 flowers onwards (M,O-P) and in stamens and petals (M-P), recapitulating the in situ hybridization pattern (J). In M, numbers indicate floral stages, the asterisk indicates the inflorescence meristem. Background fluorescence is red (M-P). (Q-T) ag-2. (Q) Inflorescence meristem, with flanking stage 2 and 3 floral primordia. (R) Late stage 4 flower. (S) Approximately stage 7 flower. (T) Late stage with several extra whorls of organs. Expression in petals. (U) Cross-section through mature flower of ag-1 mutant, with extensive signal in younger petals. (V-Y) dcl1-11. (V) Inflorescence apex (asterisk). (W) Stage 3 flower. (X) Stage 6 flower. (Y) Later stage. Interior organs develop abnormally. A-H,Q-T,V-Y were hybridized with a probe against the 3′ region of the AP2 transcript; I-L,U were hybridized with a full-length probe. Description of floral stages follows Smyth et al. (Smyth et al., 1990). se, sepal; p, petal; st, stamen; gy, gynoecium. Scale bars: 50 μm for A-L,Q-Y.

Fig. 3.

pAP3:MIM172, pAP3:amiR-AP2 and pAP3:rAP2 flowers. (A) Single flower of a pAP3:MIM172 transgenic plant; arrowhead indicates a stamen that has been partially converted into a petal, with inset showing higher magnification (see also Table S2 in the supplementary material). (B) Single flower of a pAP3:amiR-AP2 plant. Arrowhead indicates stamenoid tissue on the flanks of a petal, with inset showing higher magnification. Out of 18 T1 plants, four had flowers with slightly abnormal petals. (C) Weak pAP3:rAP2 flower. Arrowhead indicates a petaloid stamen. (D,E) Intermediate pAP3:rAP2 flowers. (F) Strong pAP3:rAP2 flower. (G) Scanning electron micrograph of a weakly affected pAP3:rAP2 flower, with perianth partially removed. (H) Higher magnification of petaloid stamen. (I) Massive organ proliferation in a single old flower of a strong line. (J) A higher magnification of I highlighting carpeloid and filamentous organs. Fractions of T1 lines in different phenotypic categories are listed in Table S2 in the supplementary material. Scale bars: 2 mm in A-F; 0.5 mm in G-J.

Fig. 4.

WUS expression in strongly affected pAP3:rAP2 flowers. (A-C) Transgenic control line containing empty vector with CaMV 35S promoter. (A) Stage 3 flower. (B) Stage 5 flower. (C) Sense probe as control. (D-I) pAP3:rAP2. (D) Stage 3 flower. (E) Stage 5 flower. (F) Approximately stage 6 flower. WUS expression persists in the center, and organ formation is delayed in the second and third whorl. (G-I) Later stages, with ectopic foci of WUS expression. Scale bars: 50 μm.

Fig. 5.

AP2 and AG expression in strongly affected pAP3:rAP2 flowers. (A-F) AP2 expression in pAP3:rAP2. (A) Stage 3 flower. (B) Late stage 3 flower. (C) Approximately stage 5 to 6 flower. (D-F) Later stages. The endogenous AP2 expression pattern (Fig. 2) was probably obscured owing to strong activity of the AP3 promoter. (G-J) AG expression in transgenic control line containing empty vector with CaMV 35S promoter. (G) Stage 2 flower. (H) Stage 3 flower. (I) Stage 6 flower. (J) Sense probe as control. Asterisk indicates the inflorescence meristem. (K-P) AG expression in strongly affected pAP3:rAP2 flowers. (K) Stage 3 flower. (L) Late stage 3 flower. (M) Approximately stage 5 to 6 flower. (N-P) Later stages. se, sepal; st, stamen; gy, gynoecium. Scale bars: 50 μm.

Fig. 6.

amiR-AG-expressing flowers. (A) Individual flowers of p35S:amiR-AG-1 plants. Out of 23 T1 plants, seven had intermediate phenotypes (left), the rest had strong phenotypes (middle and right). (B) Individual flowers of pAP3:amiR-AG-1 plants. Arrowheads indicate petaloid stamen, with higher magnification on the far right. Out of 23 T1 plants, eight had an intermediate phenotype (left), and five had a stronger phenotype (right). Scale bars: 2 mm.

Fig. 7.

Summary of interactions between A- and C-class genes. The effects on AP1 expression are inferred from previous work (Gustafson-Brown et al., 1994; Zhao et al., 2007).