Loss of LOFSEP Transcription Factor Function Converts Spikelet to Leaf-Like Structures in Rice

Abstract

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.

Figure 1.

Expression pattern analysis of rice LOFSEP genes. A to C, qRT-PCR detection of OsMADS5 (A), OsMADS1 (B), and OsMADS34 (C) transcripts. Total RNA was isolated from wild-type roots, leaves, stems, sterile lemmas, lemmas, paleas, lodicules, anthers, and pistils at stage In8 to In9 and also from 0.3- to 7-mm inflorescences. The inflorescence length around 0.3- to 0.6-mm covers the stages of In3 and In4 when the primary branches form and elongate, while inflorescence length 0.6 to 0.9 mm corresponds to stage In5 when the primary branch meristem produces secondary branches, but the SM has not yet formed. The results are presented as mean ± sd. Error bars indicate the SD for three biological replications. D to M, In situ hybridization analysis of OsMADS5 expression in the wild type at stage In3 (D), In4 (E), In5 (F), early In6 (G), Sp3 (H), Sp4 (I), Sp6 (J), Sp7 (K), and Sp8 (L), and at stage Sp6 with sense control (M). an, anther; br, bract; fm, floral meristem; im, florescence meristem; In, inflorescence; le, lemma; lo, lodicule; lsm, lateral spikelet meristem; pa, palea; PB, primary branch; pbm, primary branch meristem; pi, pistil; rg, rudimentary glume; SB, secondary branch; sbm, secondary branch meristem; sl, sterile lemma; tsm, terminal spikelet meristem. Bars = 100 µm (D–G) and 50 µm (H–M).

Figure 2.

Phenotypes of wild type, osmads5-3, osmads34-1, and osmads5-3 osmads34-1 mutants. A, E, I, and M, Spikelets of wild type (A), osmads5-3 (E), osmads34-1 (I), and osmads5-3 osmads34-1 (M) at stage In9. B, F, J, and N, Lemma and palea were removed in wild type (B) and osmads5-3 osmads34-1 (N) and half the lemma and palea were removed in osmads5-3 (F) and osmads34-1 (J) to show the inner floral organs of A, E, I, and M, respectively. C, G, K, and O, Transverse section of wild type (C), osmads5-3 (G), osmads34-1 (K), and osmads5-3 osmads34-1 (O) at stage In9 showing the identity change of sterile lemma and defects of inner floral organs in the mutants. Black stars mark the number of vascular bundles. D, H, L, and P, Diagrammatic representation of wild-type (D), osmads5-3 (H), osmads34-1 (L), and osmads5-3 osmads34-1 (P) spikelets. Q to Z, SEM analysis of the abaxial epidermis of wild-type sterile lemma (Q), lemma (R), palea (T), marginal region of palea (mrp) (U), lodicule (V), leaf blade (W), adaxial surface of wild type lemma (S), abaxial epidermis of lemma/palea-like sterile lemma of osmads34-1 (X), and osmads5-3 osmads34-1 (Y), and the abnormal elongated lodicule of osmads5-3 osmads34-1 with an mrp-like structure (Z). Black arrowheads indicate the stomata. elo, elongated lodicule; le, lemma; lo, lodicule; lsl, lemma/palea-like sterile lemma; m5, osmads5-3; m34, osmads34-1; pa, palea; pi, pistil; sl, sterile lemma; st, stamen; sti, stigma. Bars = 2 mm (A, B, E, F, I, J, M, and N), 200 µm (C, G, K, and O), 50 µm (R, S, T, U, W, and Y), and 10 µm (Q, V, X, Z).

Figure 3.

Phenotypes of osmads1-z and osmads1-z osmads5-3 mutants. A and H, Spikelets of osmads1-z (A) and osmads1-z osmads5-3 (H) at stage In9. B to D and I to K, The whole lemma and palea were removed in osmads1-z (C) and osmads1-z osmads5-3 (J) spikelets with type II phenotype; half the lemma, palea, and additional lemma/palea-like organs were removed in osmads1-z (B) and osmads1-z osmads5-3 (I) spikelets with type I phenotype; and half the lemma and palea were removed in osmads1-z (D) and osmads1-z osmads5-3 (K) spikelets with type III phenotype to show the inner floral organs at stage In9. E, F, L, and M, Transverse section of osmads1-z with type I phenotype (E) and type II phenotype (F) and osmads1-z osmads5-3 with type I phenotype (L) and type II phenotype (M) at stage In9 showing the defects in spikelets. G and N, Diagrammatic representations of osmads1-z (G) and osmads1-z osmads5-3 (N) spikelets. alo, additional lemma/palea-like organ; glo, extra glume-like second whorl organ; le, lemma; lo, lodicule; m1, osmads1-z; m5, osmads5-3; pa, palea; pi, pistil; sl, sterile lemma; st, stamen; sti, stigma. Bars = 2 mm (A–D and H–K) and 200 µm (E, F, M, and N).

Figure 4.

Phenotypes of osmads1-z osmads34-1 and osmads1-z osmads5-3 osmads34-1 mutants. A and B, Spikelets of osmads1-z osmads34-1 (A) and osmads1-z osmads5-3 osmads34-1 (B) at stage In9. C to G, Elongated leaf-like sterile lemma, lemma and palea were removed in osmads1-z osmads34-1 (C) and osmads1-z osmads5-3 osmads34-1 (D–G) to show the inner floral organs at stage In9. G1, A close-up view of the additional floral meristems marked by white arrowheads in G. G2, SEM analysis of the additional floral meristems marked by white arrowheads in G. H, All the leaf-like sterile lemmas, lemmas and paleas which were dissected from the same spikelet shown in G to show the increased number of outer spikelet organs in osmads1-z osmads5-3 osmads34-1. I, K, and M, Transverse section of osmads1-z osmads34-1 (I) and osmads1-z osmads5-3 osmads34-1 (K and M) at stage In9. Black stars mark the number of vascular bundles. J, L, and N, Diagrammatic representations of osmads1-z osmads34-1 (J) and osmads1-z osmads5-3 osmads34-1 (L and M) spikelets. O to S, SEM analysis of the abaxial epidermis of osmads1-z osmads34-1 leaf-like sterile lemma (O), leaf-like lemma/palea (Q), and a mixed epidermal pattern of extra second whorl organs consisting of leaf-like (left side), lemma/palea-like (middle), and a marginal region of the palea-like (mrp-like) structures (right side) (S), and the inner/adaxial surface of leaf-like sterile lemma (P) and lemma/palea (R). T to X, SEM analysis of the abaxial epidermis of osmads1-z osmads5-3 osmads34-1 leaf-like sterile lemma (T), leaf-like lemma/palea (V), mrp-like extra lodicules (X), and the adaxial surface of leaf-like sterile lemma (U) and lemma/palea (W). Black arrowheads indicate the stomata. af, additional floral meristems; ll, leaf-like lemma/palea; lle, leaf-like lemma; llo, leaf/glume/mrp-like second whorl organ; lpa, leaf-like palea; lsl, leaf-like sterile lemma; m1, osmads1-z; m5, osmads5-3; m34, osmads34-1; pi, pistil; st, stamen; sti, stigma. Bars = 2 mm (A–H), 1 mm (G1), 200 µm (I, K, and M), 50 µm (G2, O, Q, S, T, V, and X), and 20 µm (P, R, U, and W).

Figure 5.

Expression analysis of some floral homeotic genes in the young panicles of lofsep mutants and the wild-type backgrounds. A to J, Expression levels of class A (OsMADS14 and OsMADS15) (A and B), class B (OsMADS4 and OsMADS16) (C and D), class C (OsMADS3 and OsMADS58) (E and F), AGL6-like (OsMADS6 and OsMADS17) (G and H), and SEP3-like (OsMADS7 and OsMADS8) (I and J) genes were detected by qRT-PCR. Total RNA was isolated from 2- to 7-mm young inflorescences of the mutants of osmads1-z, osmads5-3, osmads34-1, osmads1-z osmads5-3, osmads5-3 osmads34-1, osmads1-z osmads34-1, and osmads1-z osmads5-3 osmads34-1 and also the wild-type parents Zhonghua11 and 9522. The results are presented as mean ± sd. The error bars indicate the SD for three biological replications. In, inflorescence; m1, osmads1-z; m5, osmads5-3; m34, osmads34-1; ZH11, Zhonghua 11.

Figure 6.

Expression analysis of LOFSEP genes in young panicles of lofsep mutants and wild-type backgrounds. A to D, Expression levels of LOFSEP genes OsMADS1 (A), OsMADS34 (B), and OsMADS5 (C and D) were detected by qRT-PCR. The second and third primer pairs shown in Supplemental Figure S1A were used to quantify OsMADS5 transcripts upstream (C) and downstream (D) of the T-DNA insertion site, which causes the osmads5-3 mutant, respectively. Total RNA was isolated from 2- to 7-mm young inflorescences from lofsep mutants and their wild-type backgrounds. The results are presented as mean ± sd. The error bars indicate the SD for three biological replications. In, inflorescence; m1, osmads1-z; m5, osmads5-3; m34, osmads34-1; ZH11, Zhonghua 11.

Figure 7.

Interaction analysis of OsMADS5 and OsMADS34 with other floral homeotic MADS-box proteins. Yeast two-hybrid assay shows the interaction patterns of rice LOFSEP members OsMADS5 and OsMADS34 with class A (OsMADS14 and OsMADS15), class B (OsMADS2, OsMADS4 and OsMADS16), class C (OsMADS3 and OsMADS58), class D (OsMADS13), AGL6-like (OsMADS6), SEP3-like (OsMADS7 and OsMADS8), and LOFSEP-like (OsMADS1, OsMADS5, and OsMADS34) proteins, respectively. The transformants were grown on selective Minimal Synthetic Dropout (sd) media at high stringency (SD/-Ade/-His/-Leu/-Trp/X-α-Gal). Cotransformants with empty vectors pGADT7 and pGBKT7 were used as negative controls. A, adenine; H, His; L, Leu; T, Trp.

Figure 8.

A working model summarizing the roles of homeotic MADS-box genes during rice spikelet development. A, The expression domains of A-, B-, C-, D-, and E-class and AGL6-like genes in different spikelet organs. During the spikelet development, the expression of the FUL1-like gene OsMADS14 is found in the SM; later restricted to the sterile lemmas, lemma, and palea (Pelucchi et al., 2002; Preston and Kellogg, 2006, 2007); and the FUL2-like gene OsMADS15 is also expressed in the SM and continues in the sterile lemmas, lemma, palea, and lodicules (Kyozuka et al., 2000; Preston and Kellogg, 2006, 2007). The PI/GLO-like genes OsMADS2 and OsMADS4 and the AP3/DEF-like gene OsMADS16 share a common conserved expression domain in the lodicule and stamen primordia (Nagasawa et al., 2003; Yadav et al., 2007; Yun et al., 2013). In general, the AG lineage genes OsMADS3 and OsMADS58 exhibit a very similar expression profile in the stamen, carpel, and ovule primordia (Dreni et al., 2011). The expression of the AGL11 lineage gene OsMADS13 is specific in the ovule primordium (Lopez-Dee et al., 1999; Dreni et al., 2011), and yet the other AGL11 lineage gene OsMADS21 is expressed in the developing ovule integument and very weakly in stamens and carpels (Arora et al., 2007; Dreni et al., 2007; Dreni et al., 2011). The two AGL6-like genes OsMADS6 and OsMADS17 show a largely overlapping expression patterns in the FM and, later, in palea, lodicule, and ovule (Favaro et al., 2002; Pelucchi et al., 2002; Ohmori et al., 2009; Li et al., 2010). B, Genetic and physical interactions of MADS-box factors in regulating rice spikelet morphogenesis. Black arrows indicate the interactions presented in this study and gray arrows show the interactions reported in previous works (see manuscript text for references). Bop, body of palea; Ca, carpel; FD, floral meristem determinacy; Le, lemma; Lo, lodicule; Mrp, marginal region of palea; OsM, OsMADS; Ov, ovule; Pa, palea; Pi, pistil; Rg, rudimentary glume; Sl, sterile lemma; St, stamen.