Interactions between FLORAL ORGAN NUMBER4 and floral homeotic genes in regulating rice flower development

Abstract

The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.

Keywords: Floral homeotic genes; floral meristem; flower development; FON4; genetic interaction; rice.

Fig. 1.

Phenotype of fon4-2 dep. (A, B) Wild-type (WT) flower with one lemma, one palea, two lodicules, six stamens, and one pistil. (C, D) fon4-2 flower with one lemma, one palea, two lodicules, seven stamens, and four pistils. (E, F) dep flower with one lemma, one abnormal palea, two lodicules, six stamens, and one pistil. (G, H) fon4-2 dep flower with one lemma, one abnormal palea, two lodicules, six stamens, and four pistils. The lemma and palea were removed in (B, D, F, H), and arrows indicate pistils. (I) qRT–PCR analysis of OsMADS15 expression in wild-type and fon4-2 flowers at stage Sp2/Sp4, Sp5/Sp6, and Sp7/Sp8. (J–O) OsMADS15 expression pattern in wild-type (J–L) and fon4-2 (M–O) flowers at different stages of flower development. apa, abnormal palea; fm, floral meristem; gl, glume; le, lemma; lo, lodicule; pa, palea; st, stamen. Scale bars=2 mm (A, C, E, G), 1 mm (B, D, F, H), and 50 μm (J–O).

Fig. 2.

Phenotype of fon4-2 spw1-1. (A) spw1-1 flower with glume-like organs and ectopic carpels. (B) fon4-2 spw1-1 flower. The palea and lemma were removed in (A) and (B). (C) qRT–PCR analysis of OsMADS16 expression in wild-type and fon4-2 flowers. (D–I) OsMADS16 expression pattern in wild-type (D–F) and fon4-2 (G–I) flowers. ec, ectopic carpel; glo, glume-like organ; lo, lodicule; st, stamen. Scale bars=1 mm (A, B) and 50 μm (D–I).

Fig. 3.

Phenotype of fon4-2 osmads3-4. (A) osmads3-4 flower. (B) fon4-2 osmads3-4 flower. The lemma and palea were removed in (A) and (B), and arrowheads indicate lodicule-like organs. (C) A close-up of normal stamens (arrows) and lodicule-like organs (arrowheads) in (B). (D) SEM observation of numerous carpels in fon4-2 osmads3-4. (E–J) SEM images of wild-type (E), fon4-2 (F), osmads3-4 (G), and fon4-2 osmads3-4 (H–J) flowers at the stage when stamen primordia are formed in whorl 3. Arrowheads in (F–J) indicate organ primordia developed from the additional whorl. A partial palea was removed in (J). (K) SEM image of a wild-type flower at the stage when the pistil with two stigmas is produced. (L) SEM image of a fon4-2 osmads3-4 flower at the stage that corresponds to that of (K). The floral meristem (red region) remains in fon4-2 osmads3-4 even after several carpels have been formed. (M–P) OSH1 expression in wild-type (M), fon4-2 (N), osmads3-4 (O), and fon4-2 osmads3-4 (P) flowers after the formation of carpels. OSH1 expression completely disappears when the carpels are formed in the wild-type and osmads3-4 flowers, whereas it continues to be expressed in the FM around the carpel primordia in fon4-2 and fon4-2 osmads3-4 flowers. Moreover, the OSH1 expression region is larger in fon4-2 osmads3-4 than in the fon4-2 single mutant. (Q) qRT–PCR analysis of OsMADS3 expression in wild-type and fon4-2 flowers. (R–W) OsMADS3 expression pattern in wild-type (R–T) and fon4-2 (U–W) flowers. ca, carpel; ec, ectopic carpel; fm, floral meristem; ll, lodicule-like organ; st, stamen. Scale bars=1 mm (A–C), 500 μm (D), 50 μm (E–J, R–W), and 100 μm (K–P).

Fig. 4.

Phenotype of fon4-1 osmads58. (A–C) The flowers of osmads58 (A), fon4-1 (B), and fon4-1 osmads58 (C) with the removal of the lemma and half the palea. Arrows indicate pistils. (D) qRT–PCR analysis of OsMADS58 expression in wild-type and fon4-2 flowers. (E–J) OsMADS58 expression pattern in wild-type (E–G) and fon4-2 (H–J) flowers. ca, carpel; fm, floral meristem; st, stamen. Scale bars=1 mm (A–C) and 50 μm (E–J).

Fig. 5.

Phenotype of fon4-1 osmads13-3. (A, C, E) The flowers of fon4-1 (A), osmads13-3 (C), and fon4-1 osmads13-3 (E) with the removal of the lemma and half the palea. (B, D, F) Longitudinal section of fon4-1 (B), osmads13-3 (D), and fon4-1 osmads13-3 (F) stained with 0.1% Toluidine blue showing carpel and ovule development. The arrowheads in (D) and (F) indicate the carpelloid structures converted from the ovules. (G) SEM image of carpelloid structures (red region) in fon4-1 osmads13-3. (H–J) OSH1 expression in fon4-1 (H), osmads13-3 (I), and fon4-1 osmads13-3 (J). (K) qRT–PCR analysis of OsMADS13 expression in wild-type and fon4-2 flowers. (L–Q) OsMADS13 expression pattern in wild-type (L–N) and fon4-2 (O–Q) flowers. ca, carpel; fm, floral meristem; ov, ovule. Scale bars=1 mm (A, C, E), 100 μm (B, D, F, G), and 50 μm (H–J, L–Q).

Fig. 6.

Phenotype of fon4-2 osmads1-z. (A) osmads1-z flower with leaf-like lemma and palea. (B–D) osmads1-z flowers of type I (B), type II (C), and type III (D). A half of a leafy lemma and palea were removed, and arrowheads in (B) and (C) indicate glume-like organs. (E, F) Type IV flower of osmads1-z with a new spikelet in the center. The spikelet in (E) has stamens and carpels in the inner whorls, whereas the spikelet in (F) only contains repetitious glume-like organs (arrowheads). A half of a leafy lemma and palea of primary and secondary flowers were removed in (E), and the whole leafy lemma and palea were removed in (F). (G) Type I flower of fon4-2 osmads1-z generating a new indeterminate composed of only glume-like organs. (H) SEM image of that in (G). The red square indicates the indeterminate FM. (I) Type II flower of fon4-2 osmads1-z generating a new determinate spikelet with stamens and carpels in the inner whorls. (J) Longitudinal section of the spikelet in (I). (K) Type III flower of fon4-2 osmads1-z containing only a glume on the pedicel. (L) Type IV flower of fon4-2 osmads1-z with an undefined structure (red arrowhead) inside the flower. (L1) A close-up view of the undefined structure in (L). (M, N) SEM images of osmads1-z (M) and fon4-2 osmads1-z (N) at the stage when the glume, lemma, and palea primordia are formed. (O) SEM image of the osmads1-z flower at the stage when stamen primordia initiate. (P–V) SEM images of fon4-2 osmads1-z flowers. (W) qRT–PCR analysis of OsMADS1 expression in wild-type and fon4-2 flowers. (X1–Y3) OsMADS1 expression pattern in wild-type (X1–X3) and fon4-2 (Y1–Y3) flowers. Arrowheads in (X3) and (Y3) indicate carpels. sp, spikelet; fm, floral meristem; gl, glume; le, lemma; pa, palea; pe, pedicel; glo, glume-like organ; st, stamen. Scale bars=1 mm (A–G, I, K, L), 500 μm (L1), 100 μm (H, J, S–V), and 50 μm (M–R, X1–Y3).

Fig. 7.

Comparison of the expression pattern of FON4 and floral homeotic genes from stage Sp2 to Sp7. Blue indicates the expression region of FON4, and the green, red, pink, and orange oblique lines indicate the expression region of OsMADS1, OsMADS3, OsMADS58, and OsMADS13, respectively. At the stages (red square) when apparent abnormalities are observed in the double mutants, the expression region of FON4 partially overlaps with these genes in the floral meristem. fm, floral meristem; gl, glume; le, lemma; lo, lodicule; pa, palea; st, stamen.