Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy

Abstract

Reproductive organ development is one of the most important steps in the life cycle of plants. Studies using core eudicot species like thale cress (Arabidopsis thaliana) and snapdragon (Antirrhinum majus) have shown that MADS domain transcription factors belonging to the AGAMOUS (AG) subfamily regulate the identity of stamens, carpels, and ovules and that they are important for floral meristem determinacy. Here, we investigate the genetic interactions between the four rice (Oryza sativa) AG subfamily members, MADS3, MADS13, MADS21, and MADS58. Our data show that, in contrast with previous reports, MADS3 and MADS58 determine stamen and carpel identity and, together with MADS13, are important for floral meristem determinacy. In the mads3 mads58 double mutant, we observed a complete loss of reproductive organ identity and massive accumulation of lodicules in the third and fourth floral whorls. MADS21 is an AGL11 lineage gene whose expression is not restricted to ovules. Instead, its expression profile is similar to those of class C genes. However, our genetic analysis shows that MADS21 has no function in stamen, carpel, or ovule identity determination.

Figure 1.

Expression Analysis of AG Family MADS Box Genes. (A) to (E) Expression profile of MADS3 in wild-type floral buds ([A] to [D]) and in the mads3-3 mutant as negative control (E). (F) to (I) Expression profile of MADS58 in wild-type floral buds ([F] to [H]) and in the mads58 mutant as negative control (I). (J) to (N) MADS13 activation in the wild-type FM (J) and its expression in wild-type ovules (K), at the adaxial side of a mads3-3 mads58 double mutant palea-like primordium (L) and at a later developmental stage of the same mutant in the central vasculature parenchyma (M). As expected, no signal is visible in the mads3-3 mads13 mads58 triple mutant vasculature (N). (O) to (R) Comparative expression of MADS21 at similar stages of wild-type ([O] and [P]) and pMADS13:MADS21 ([Q] and [R]) ovule primordia. Arrows in (P) indicate the integument primordia. (S) to (U) DL expression in the wild-type carpel (S) and in the mads3-3 mads58 double mutant palea-like primordia ([T] and [U]). In (S) and (T), the expression is clearly visible also in the lemma midrib. The early palea-like primordium (T) and the palea-like vasculature (U) are indicated with an arrowhead and an arrow, respectively. There is no obvious expression in the vasculature. (V) DL expression in the mads3-3 mads13 mads58 triple mutant palea-like primordium; no expression is observed in the differentiating vascular region (arrows). (W) and (X) G1 expression profile. (W) Expression in the wild-type sterile glumes (arrowheads) and palea (arrow). (X) Expression in the mads3-3 mads58 double mutant floral apex during the formation of the fourth whorl. c, carpel; elo, ectopic lodicule; fm, floral meristem; l, lemma; lo, lodicule; p, palea; s, stamen. Bars = 50 μm.

Figure 2.

Rice AG Family Gene Mutant Phenotypes. (A) Wild-type mature rice flower. (B) Mads3-4 mutant flower. The arrow indicates the more severely affected palea-side stamen. (C) Mild mads3-3 mutant phenotype. The arrow indicates the third-whorl ectopic lodicule replacing the palea-side stamen. (D) Severe mads3-3 mutant phenotype. (E) Mads3-3/+ mads21 mads58 mutant flower showing no phenotype. (F) Mads3-3 mads58/+ flower. (G) Mads3-3 mads58 double mutant. A palea-like organ developed in place of the carpel toward the lemma side (arrow). (H) Mads3-4 mads58 double mutant. Rarely, like in this picture, the palea-like organ does not develop at the lemma side (arrow). (I) Mads3-3 mads13 mads58 triple mutant showing an increased development of the palea-like organ. (J) and (K) Mads3-3 mads13 (J) and mads13 mads58 (K) double mutants. (L) Mads3-3 mads13/+ mads58/+ flower. After producing a few ectopic carpelloid structures, the indeterminate FM switched to the differentiation of ectopic lodicule primordia (arrow). To show the inner whorls, lemma and palea were partially or completely removed. In all of the pictures, the asterisk marks the second whorl lodicule, whereas the arrowheads in (G), (J), and (L) indicate the visible indeterminate FM. Bars = 100 μm.

Figure 3.

Scanning Electron Microscopy Analysis of Mutant Flowers. (A) Inner organs of a mads3-3 mads58 double mutant floral bud showing a second-whorl lodicule (asterisk), a fourth-whorl lemma-side palea-like primordium (arrow), several ectopic lodicules, and the enlarged FM. (B) Wild-type sterile lemma with the abaxial surface oriented on the left. (C) to (F) Abaxial surface of wild-type lemma (C), wild-type palea (D), mads3-3 mads58 double mutant palea-like organ (E), and mads3-3 mads13 mads58 triple mutant palea-like organ (F). The palea and palea-like marginal regions are shown in (D) to (F) (arrows). (G) to (K) Adaxial surface of wild-type lemma (G), wild-type palea (H), mads3-3 mads58 double mutant palea-like organ ([I] and [J]), and mads3-3 mads13 mads58 triple mutant palea-like organ (K). (L) Mads3-3 mads13 double mutant indeterminate FM surrounded by several developing carpel primordia and also an ectopic lodicule primordium (arrow). Bars = 100 μm in (D) and 50 μm in the other pictures.

Figure 4.

Whole-Mount Tissue Clearing Analysis. (A) and (D) Wild-type mature ovule showing details (D) of the central cell nuclei (arrow), the egg cell nucleus (arrowhead), and the inner integument, which is laterally ~4 μm thick (bracket). (B) and (E) mads13/pMADS13:MADS21 mutant ovule showing details (E) of the tracheary element replacing the embryo sac (arrow) and the inner integument, which is laterally ~16 μm thick (bracket). (C) and (F) A mads13 mutant ovary that is filled with carpelloid cells. ov, ovary wall. Bars = 50 μm for (A) to (C) and 20 μm for (D) to (F).

Figure 5.

Genetic Model for Floral Organ Identity Determination in Rice. The existence of a true rice A function determining the identity of the first- and second-whorl organs has, like in most flowering plants, not been identified yet. The homeotic conversion of the carpel into a palea-like organ in the mads3 mads58 double mutant suggests that only the palea, and not the lemma, can be considered as a first-whorl organ. The class B genes regulate lodicule identity in the second whorl, and together with class C genes, they determine stamen identity in the third whorl. In the fourth whorl, DL represses the expression of class B genes. The main class C genes of rice are MADS3 and MADS58. Since the ovule develops directly from the FM, it can be considered a fifth-whorl organ. Based on current data, MADS13 seems to be the only AG subfamily gene regulating ovule identity in rice. Among the four AG subfamily genes of rice, MADS3, MADS13, and MADS58 regulate also FM determinacy, whereas MADS21 seems not to play important functions during flower development. The MADS domain proteins shown in this model might interact with SEP-like and/or AGL6-like MADS domain proteins providing E-function (Favaro et al., 2002; Kater et al., 2006; Ohmori et al., 2009; Cui et al., 2010; Li et al., 2010; Li et al., 2011a), which for simplicity are not shown in this scheme.