leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development

Abstract

Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 (lhs1). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1-conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene.

Figure 1.

Construction of Truncated OsMADS1 and Phenotype of Transgenic Plants. (A) Schematic representations of truncated OsMADS1 proteins, phenotype, and RNA gel blot analyses of the transgenic plants. Shown at left is the OsMADS1 protein represented by four regions: M, MADS domain; I, I region; K, K domain; C, C-terminal region. The numbers indicate the boundaries of the regions and the positions of the truncations. In the center is the phenotype of the T2 transgenic rice plants that express the construct given at left. Shown at right are RNA gel blot data from the independent transgenic plants, which were derived from transformation of the constructs shown at left. Equal amounts of total RNA loading were examined by using ethidium bromide staining of 25S and 18S rRNAs (data not shown). In pGA1511-2 transgenic lines, line 1 flowered ∼10 days earlier than wild-type or control plants carrying the binary vector pGA1671; the other lines flowered ∼5 days earlier than controls. In pGA1858, pGA1857, and pGA1856 transgenic lines, lines 1858-4, 1858-5, 1857-1, 1857-2, 1857-5, 1856-2, 1856-3, 1856-4, and 1856-5 flowered ∼5 days earlier than controls, and other lines did not show early flowering. Five micrograms of total RNA from prepared leaves was loaded in all other lanes. The C-terminal region of OsMADS1 was used as a gene-specific probe (Chung et al., 1994), except that RNAs from pGA1856 lines were hybridized with the K region of OsMADS1. W, wild-type plants; numbers, independent transgenic lines. (B) Phenotypes of transgenic plants expressing full-length OsMADS1 or the C-terminal truncated form. Shown at left is a control plant transformed with the binary vector pGA1671; at center is the transgenic plant with pGA1511-2 (line 1511-2-1); at right is the transgenic plant with pGA1856 (line 1856-2). (C) Spikelets of the wild type (left) and transgenic line 1511-2-1 (right). Normal glumes are short and inconspicuous (arrowheads), whereas the transgenic spikelet shows overgrowth of glumes (arrows) that resemble paleae and lemmas. This phenotype was observed in most transgenic plants expressing the full-length or the C-terminal–truncated OsMADS1.

Figure 2.

MADS Domain Sequences of the Site-Directed OsMADS1 Mutants. At left are the amino acids that differ from those of the wild-type MADS domain sequence. The SRF MADS domain region and the amino acid residues involved in DNA contacting or dimerization are shown. The asterisk indicates two amino acid residues generated in the place of Leu⁸. At right are the binary vectors, which consist of the actin act1 promoter and the mutant OsMADS1.

Figure 3.

Phenotypes of Transgenic Plants Expressing the OsAMDS1 Mutant. (A) A spikelet from a plant of transgenic line 1703-1 transformed with pGA1703. The spikelet has a phenotype similar to lhs1, showing an open flower with the elongated leafy palea and lemma. (B) A spikelet of a wild-type plant. (C) A cross-sectioned spikelet from a plant of line 1703-1. The spikelet consists of normal glumes, abnormal palea and lemma, two pairs of leafy palea- and lemma-like lodicules, four stamens, and a carpel. (D) A cross-sectioned wild-type spikelet. The spikelet has glumes, palea and lemma, a pair of lodicules, six stamens, and a carpel. (E) A spikelet from the line 1703-3. The spikelet has an additional palea- and lemma-like structure (arrowhead). Original palea and lemma developed abnormally. Other organs—lodicules, stamens, and a carpel—are almost identical to the wild type. (F) RNA gel blot analysis of the OsMADS1 transcript in the leaves and flowers of the 1703 transgenic lines. Five micrograms of total RNA from leaves (left) and 10 μg from flowers (right) were used for RNA gel blot analysis. At top, the amount of OsMADS1 transcript expressed was measured using the gene-specific probe for OsMADS1. Because of the similar sizes of the endogenous and transgenic OsMADS1 transcripts, the signals shown are the sum of the two. Lanes W, wild type; lanes 1, line 1703-1; lanes 2, line 1703-2; lanes 3, line 1703-3. At bottom are the controls. The same filters were washed and rehybridized with the rice α-tubulin gene OsTubA1 (Jeon et al., 2000). c, carpel; g, normal glume; l, abnormal lemma; lo, leafy palea- and lemma-like lodicules; p, abnormal palea; s, stamen.

Figure 4.

Amino Acid Changes Corresponding to Nucleotide Changes in the lhs1 Allele. The amino acid sequence is in the MADS box domain. Boldface letters indicate the changes in the lhs1 allele.

Figure 5.

Structure of the OsMADS1 Genomic Clone, the Vector Used for Genetic Complementation, and Phenotypes of the Transgenic Plants. (A) A restriction map of the OsMADS1 genomic clone is shown at top. B, BamHI; K, KpnI; N, NotI; R, EcoRI; S, SacI; V, EcoRV; X, XbaI; Xh, XhoI. At center is the genomic structure of OsMADS1. The 11,851-bp EcoRI fragment contains the entire OsMADS1 gene, which consists of seven introns (white bars) and eight exons (black bars) (GenBank accession number AF204063). The length of each intron is, in order from left, 5524, 771, 120, 207, 90, 218, and 378 bp. The numbers on top of the DNA fragment indicate the introns. The promoter and terminator regions are shown in the diagonally striped rectangles. The open arrow indicates the direction of transcription. At bottom is the binary vector pGA2122 containing the OsMADS1 genomic clone. The vector also harbors the hygromycin phosphotransferase (hph) gene under the control of the 35S promoter for selection of transgenic plants. (B) A spikelet from a transgenic plant from line 2122-4. The structure of palea and lemma in plant 2122-4 is indistinguishable from that of a wild-type spikelet. (C) A dissected spikelet from a plant of line 2122-4 showing inner floral organs. The spikelet consists of a pair of lodicules, six stamens, and a carpel, which are identical to those of the wild type. (D) A spikelet from a transgenic plant from line 2122-6. The palea is partially recovered. c, carpel; g, normal glume; l, abnormal lemma; lo, leafy palea- and lemma-like lodicules; p, abnormal palea; s, stamen.

Figure 6.

Spikelets from lhs1 and Transgenic Plants. (A) Panicles from wild-type (left) and lhs1 (right) plants. (B) A spikelet from the lhs1 mutant. The palea and lemma are overdeveloped in comparison with those of the wild type (see Figure 3B). The spikelet is open because of abnormal growth of leafy palea and lemma. (C) A dissected lhs1 spikelet in which a palea and lemma were ripped off. (D) An lhs1 spikelet generating a new floret. (E) An lhs1 spikelet generating a new flower (arrowhead) consisting of leafy palea and lemma. (F) An lhs1 spikelet generating a new flower in sequence. (G) A transgenic spikelet from transgenic line 2145-9 generating multiple paleae and lemmas (arrowhead). (H) A cross-section of an lhs1 spikelet. The spikelet consists of normal glumes, abnormal leafy palea and lemma, two pairs of leafy palea- and lemma-like lodicules, four stamens, and a carpel. (I) A cross-section of an lhs1 spikelet. The spikelet consists of leafy palea and lemma, abnormal lodicules, six stamens, and two carpels. (J) A cross-section of an lhs1 spikelet. The spikelet consists of two pairs of lodicules, eight stamens, and two carpels. (K) A cross-section of a spikelet from a plant of transgenic line 2145-8. (L) A cross-section of anthers of a spikelet from a plant of line 2145-8. (M) A cross-section of a spikelet from a plant of line 2145-9, showing multiple paleae and lemmas. a, anther; c, carpel; f, flower; g, glume; l, lemma; lo, lodicule; p, palea; s, stamen; st, stigma.

Figure 7.

Scanning Electron Microscopy of Wild-Type, lhs1, and Transgenic Spikelets. Scanning electron microscopy of spikelets of wild-type ([A] to [G]), lhs ([H] to [O]), and transgenic line 2145-9 (P) rice. (A) In outer whorls, glumes and palea and lemma primordia are formed. The central floral meristem starts initiating stamen primordia. . (B) Six developing stamens are visible. One stamen primordium is developing late (arrowhead). Removal of the lemma reveals that lodicule primordia form at this stage (data not shown). . (C) The carpel primordium is apparent. . (D) A dissected spikelet in which palea and lemma were ripped off. A pair of lodicules is formed between stamens and a lemma. . (E) In stamens, filaments and anthers are apparently differentiated. . (F) The mature spikelet in which palea and lemma were ripped off. . (G) The palea and lemma of a mature spikelet. . (H) Glume and palea and lemma primordia are formed in outer whorls of a lhs spikelet. In the inner whorls, a new palea- and lemma-like structure is visible. . (I) Palea- and lemma-like structures and a floral meristem are formed in the inner whorls of the lhs1 spikelet. The central meristem is differentiated to stamens and a carpel. . (J) Top view of (I). . (K) The lhs spikelet carries five stamens. Lodicules are formed at the base of the stamens. . (L) The spikelet consists of leafy lodicules, three stamens, and two carpels. Palea, lemma, and right lodicule were removed. . (M) The spikelet consists of leafy lodicules, two stamens, and a new flower. Palea and lemma were removed to reveal inner organs. (N) A mature spikelet. Because of the leafy characteristic of the palea and lemma, the spikelet is open. . (O) The spikelet exhibits successive formation of paleae and lemmas (arrowhead). . (P) Early development of the spikelet from a plant of transgenic line 2145-9. The young spikelet consisted of successive leafy paleae and lemmas and floral primordia. . c, carpel; fm, floral primordium; g, glume; l, lemma; lo, lodicule; p, palea; pl, palea- and lemma-like structure; s, stamen; t, trichome.