Regulatory Role of OsMADS34 in the Determination of Glumes Fate, Grain Yield, and Quality in Rice

Abstract

Grasses produce seeds on spikelets, a unique type of inflorescence. Despite the importance of grass crops for food, the genetic mechanisms that control spikelet development remain poorly understood. In this study, we used m34-z, a new mutant allele of the rice (Oryza sativa) E-class gene OsMADS34, to examine OsMADS34 function in determining the identities of glumes (rudimentary glume and sterile lemma) and grain size. In the m34-z mutant, both the rudimentary glume and sterile lemma were homeotically converted to the lemma-like organ and acquired the lemma identity, suggesting that OsMADS34 plays important roles in the development of glumes. In the m34-z mutant, most of the grains from the secondary panicle branches (spb) were decreased in size, compared with grains from wild-type, but no differences were observed in the grains from the primary panicle branches. The amylose content and gel consistency, and a seed-setting rate from the spb were reduced in the m34-z mutant. Interesting, transcriptional activity analysis revealed that OsMADS34 protein was a transcription repressor and it may influence grain yield by suppressing the expressions of BG1, GW8, GW2, and GL7 in the m34-z mutant. These findings revealed that OsMADS34 largely affects grain yield by affecting the size of grains from the secondary branches.

Keywords: OsMADS34; grain size; rice (Oryza sativa L.); rudimentary glume; spikelet; sterile lemma; transcriptional repressor.

FIGURE 1

Phenotypes of spikelets in the wild-type and the m34-z mutant. (A) Wild-type spikelet. (B) Partial magnification in (A). (C) Wild-type floret. (D) Histological analysis of the wild-type floret. (E) Histological analysis of the sterile lemma in the wild-type spikelet. (F) Histological analysis of the rudimentary glume in the wild-type spikelet. (G) m34-z mutant spikelet. (H) Partial magnification in (G). (I) m34-z mutant floret. (J) Histological analysis of the m34-z mutant floret. (K) Histological analysis of the sterile lemma in the m34-z mutant spikelet. (L) Histological analysis of the rudimentary glume in the m34-z mutant spikelet. rg, rudimentary glume; sl, sterile lemma; le, lemma; pa, palea; lo, lodicule; st, stamen; pi, pistil. Red stars represent vascular bundles. Bars = 1000 μm in (A,B,G), and H; 500 μm in (C,I); and 100 μm in (D–F,J–L).

FIGURE 2

scanning electron microscopy (SEM) analysis of glumes in the wild-type and the m34-z mutant at heading stage. (A–E) Wild-type spikelet. (B) Epidermal surface of the wild-type lemma. (C) Epidermal surface of the wild-type palea. (D) Epidermal surface of the wild-type sterile lemma. (E) Epidermal surface of the wild-type rudimentary glume. (F–H) m34-z mutant spikelet. (G) Epidermal surface of the m34-z mutant sterile lemma. (H) Epidermal surface of the m34-z mutant rudimentary glume. rg, rudimentary glume; sl, sterile lemma; le, lemma; pa, palea; bop, body of palea; mrp, marginal region of palea. Bars = 1000 μm in (A,F) and 100 μm in (B–E,G–H).

FIGURE 3

Relative expression levels of genes involved in floral organ development in the wild-type and the m34-z mutant spikelet. rg, rudimentary glume; sl, sterile lemma; le, lemma; pa, palea; WT, wild-type. Error bars indicate SD. ^∗∗Significant difference at P < 0.01 compared with the wild-type by Student’s test.

FIGURE 4

Spikelets at early developmental stages in the wild-type and the m34-z mutant. (A–E) wild-type spikelet. (A) Sp4, (B) Sp5-6, (C) Sp7, (D) Sp8, (E) After Sp8, (F–I) m34-z mutant spikelet, (F) Sp4, (G) Sp5-6, (H) Sp7, (I) Sp8, (J) After Sp8. fm, floral meristem; rg, rudimentary glume; sl, sterile lemma; le, lemma; pa, palea; st, stamen; pi, pistil. Bars = 100 μm.

FIGURE 5

Phenotypic observations of grain and pollen viability in the wild-type and the m34-z mutant. (A–E) Phenotypes of the grain and pollen viability from the primary panicle branches (ppb) in the wild-type. (F–J) Phenotypes of the grain and pollen viability from the ppb in the m34-z mutant. (K–O) Phenotypes of the grain and pollen viability from the secondary panicle branches (spb) in the wild-type. (P–T) Phenotypes of the grain and pollen viability from the spb in the m34-z mutant. (A,F,K,P) grain size. (B,G,L,Q) size of brown rice. (C–D,H–I,M–N,R–S) epidermal surface of brown rice. E, J, O, and T, pollen viability. Bars = 1000 μm in (A–C,F–H,K–M), and P-R; 50 μm in (D,I,N,S); 100 μm in (E,J,O,T).

FIGURE 6

OsMADS34 influences cell expansion. (A) Inner epidermal cells of hulls from the ppb in the wild-type. (B) Inner epidermal cells of hulls from the ppb in the m34-z mutant. (C) Inner epidermal cells of hulls from the spb in the wild-type. (D) Inner epidermal cells of hulls from the spb in the m34-z mutant. (E) Cell length. (F) Cell width. (G) Grain from the ppb in the wild-type. (H) Grain from the ppb in the m34-z mutant. (I) Grain from the spb in the wild-type. (J) Grain from the spb in the m34-z mutant. (K) Total cell number along the longitudinal axis of the lemma. (L) Total cell number per millimeter along the longitudinal axis of the lemma. (M) Expression analysis of cell expansion-related genes in young panicles. WT-p, grains from the ppb in the wild-type; WT-s, grains from the spb in the wild-type; m34-z-p, grains from the ppb in the m34-z mutant; m34-z-s, grains from the spb in the m34-z mutant. The red boxes indicate the middle part of the lemma in (G,H,I,J). Bars = 50 μm in (A–D). Error bars indicate SD. ^∗∗Significant difference at P < 0.01 compared with the wild-type by Student’s test.

FIGURE 7

Identification of OsMADS34 gene as the causal gene for the the m34-z mutant. (A–C) Map position of the OsMADS34 locus. The relative position of the BAC clone is shown. Genomic structure of OsMADS34. The mutated site of the m34-z mutant is shown. (D) OsMADS34 encodes a 239 amino acid protein, while the mutation of OsMADS34 in the m34-z mutant encodes a 76 amino acid protein due to a coding frame-shift. (E–K) Complementation test. All mutant phenotypes (deformed spikelets, reduced grains, and abnormal epidermal surface) were rescued in the transgenic plants. (E) Deformed spikelets were recovered in the rescued lines. (F,H) Grains from the spb in the m34-z mutant. (G,I) Reduced grains from the spb in the m34-z mutant were rescued in the transgenic lines. (J) Epidermal surface of brown rice from the spb in the m34-z mutant. (K) Epidermal surface of brown rice from the spb in the m34-z mutant was similar with that of the wild-type in the transgenic lines. Bars = 1 cm in (E); 1000 μm in (F–I); 50 μm in (J–K).

FIGURE 8

Subcellular localization of the OsMADS34 protein. (A–C) GFP fusion protein. (A) differential interference contrast (DIC) image; (B) bright-field image; (C) merged image of GFP fusion protein. (D–F) AFD1-GFP. (D) DIC image; (E) bright-field image; (F) merged image of AFD1-GFP fusion protein. (G–I) OsMADS34-GFP. (G) DIC image; (H) bright-field image; (I) merged image of OsMADS34-GFP fusion protein. Bars = 5 μm in (B–G).

FIGURE 9

Expression patterns and transcriptional activity analysis. (A) OsMADS34 expression in different tissues detected by qPCR. (B) GUS staining of root. (C) GUS staining of culm. (D) GUS staining of leaf. (E) GUS staining of spikelet. (F,G) Transcriptional activity test. R, root; C, culm; L, leaf; Rg, rudimentary glume; Sl, sterile lemma; Le, lemma; Pa, palea; Lo, lodicule; st, stamen; pi, pistil; 0.5 cm, young panicles (≤0.5 cm); 0.5–2 cm, young panicles (0.5–2 cm); 2–5 cm, young panicles (2–5 cm); 1D, 1 day after pollination; 4D, 4 days after pollination; 7D, 7 days after pollination; 10D, 10 days after pollination. Bars = 1 cm in (B–G). Error bars indicate SD. ^∗∗Significant difference at P < 0.01 compared with the wild-type by Student’s test.

FIGURE 10

Expression of genes related to glume development at different stages. 0.5 cm, young panicles (≤0.5 cm); 0.5–2 cm, young panicles (0.5–2 cm); 2–5 cm, young panicles (2–5 cm). Error bars indicate SD. **Significant difference at P < 0.01 compared with the wild-type by Student’s test; *Significant difference at P < 0.05 compared with the wild-type by Student’s test.