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doi: 10.1242/10.1242/dev.00294

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SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

Nobuhiro Nagasawa^1,4, Masahiro Miyoshi^1,5, Yoshio Sano², Hikaru Satoh³, Hiroyuki Hirano¹, Hajime Sakai^4, and Yasuo Nagato^1,

¹ Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
² Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
³ Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
⁴ DuPont Company, Agriculture and Nutrition, Delaware Technology Park 200, 1 Innovation Way, Newark, DE 19714, USA
⁵ Present address: Orynova Co. Ltd., Iwata 438-0802, Japan

View larger version (51K): [in a new window]   Fig. 1. Phenotypes of wild-type, spw 1-1, dl-sup 1 and dl-sup 1 spw 1-1 flowers. (A) A wild-type flower with two empty glumes (eg), lemma (le), palea (pa), two lodicules (lo), six stamens (st) and one gynoecium (arrowhead) with two stigmas (sti). A half of the lemma and palea were removed to show the inside of the flower. (B) A spw 1-1 flower. Stamens and lodicules are homeotically transformed into carpels (arrowheads) and palea-like organs (arrows), respectively. (C) A dl-sup 1 flower. The carpel is homeotically transformed into ectopic stamens (arrowheads). (D) A spw 1-1 dl-sup 1 flower with ectopic palea-like organs (arrowheads) and organs of unknown identity (arrows). The lemma and a half of palea were removed in B-D. (E-H) Floral diagram of (E) the wild-type flower, (F) the spw 1-1 flower, (G) the dl-sup 1 flower, and (H) the dl-sup 1 spw 1-1 flower. ra, rachilla. Bar: 1 mm.

View larger version (117K): [in a new window]   Fig. 2. Micrographs of spw1-1 flowers. (A) A wild-type palea with a membranous marginal region (left) and the lemma with trichomes on the entire surface (right). (B) Scanning electron micrographs (SEM) of the abaxial, marginal region of the wild-type palea. (C) SEM of the abaxial surface of a palea-like organ of the spw1-1 flower. (D) The spw1-1 flower with three palea-like organs (arrowheads) and one carpeloid palea-like organ (arrow). (E) Apical portion of the carpeloid palea-like organ with stigmatic cells (arrow). (F) A transverse section of the spw1-1 flower with seven carpels (arrowheads) and three palea-like organs (arrows). (G) A side view of the spw1-1 flower with nucellar tissues protruding from the ovary (arrowhead). (H) A longitudinal section of the spw1-1 ovary with nucellus and degenerated embryo sac (arrowhead). (I) A transverse section of nucellar tissues of an ectopic carpel formed in whorl 3, which is completely protruding from the spw1-1 ovary without internal differentiation. nu; nucellar tissue. Bar, 1 mm in (D,F,G) and 30 µm in (A-C,E,H,I).

View larger version (178K): [in a new window]   Fig. 3. Scanning electron micrographs of early developmental stages of spw1-1, dl-sup1 and dl-sup2 flowers. (A-C) Wild-type; (D-F) spw1-1; (G-I) dl-sup1; (J-L) dl-sup2. (A,D,G,J) Young flowers at the stage when organ primordia were formed in whorl 3. (B,E,H,K) Young flowers at the stage when stamen primordia have started to form the anther structure in wild type. (C,F,I,L) Flowers at the later stage when the carpel primordia start to form stigmas in wild type. (D,G,J) In spw1-1, dl-sup1 and dl-sup2 flowers, six organ primordia were formed at the positions where stamen primordia were formed in the wild type. (E) In spw1-1, six organ primordia in whorl 3 broaden to form carpels. (F) Emergence of ectopic palea-like organ primordia in spw1-1. (H,K) dl-sup1 and dl-sup2 flowers. The primordium of the first ectopic stamen (arrowhead) is developing from the lemma side of whorl 4. (I,L) At the later stage, the floral apical meristem (arrowhead) has continued to produce many ectopic stamens. le, lemma; pa, palea; ep, ectopic palea-like organ; es, ectopic stamen. Bars: 30 µm.

View larger version (69K): [in a new window]   Fig. 4. Phenotype of dl-1(T65) plants in the vegetative phase. (A) Wild-type plant in the vegetative phase. (B) dl-1(T65) plant at the same stage with drooping leaves. (C) Transverse section of the wild-type leaf blade. The midrib (MR) in the middle of the leaf blade has large clear cells (arrowhead), and an overall structure that is distinct from that of the lateral veins (arrow). (D) Transverse section of the dl-1(T65) leaf blade that lacks a midrib. The vein in the middle of the leaf blade (arrowhead) resembles the lateral veins (arrow). Bars (C,D), 200 µm.

View larger version (133K): [in a new window]   Fig. 5. Micrographs of wild-type and dl flowers. The lemma and a half of palea were removed in all flowers. (A) A wild-type flower with two empty glumes, two lodicules, six stamens and one carpel with two stigmas. (B) A dl-1(H0788) flower forming the carpel with three stigmas (arrowheads). (C) A dl-1(T65) flower with an abnormal cell mass produced from the carpel (arrowhead) and an ectopic stamen (arrow) emerging from the base of the carpel. (D) A dl-1(T65) flower producing an extra stamen (arrow) and a mosaic organ, staminoid carpel (arrowhead), in which an anther is formed on the ovary. (E) A dl-2 flower without any apparent abnormality. (F) A dl-sup1 flower with a homeotic conversion of the carpel to stamens. (G) A cross section of the dl-sup1 flower. In addition to the six normal stamens (arrowheads) and two lodicules, ectopic stamens (arrows) are formed. (H) Extra stamens (arrowheads) are alternately produced in the position of the carpel in dl-sup1 flowers. (I) A dl-sup2 flower with homeotic conversion of the carpel into stamens. Bars: 500 µm.

View larger version (79K): [in a new window]   Fig. 6. Micrographs of the spw1-1 dl-sup1 double mutant. (A) A spw1 dl-sup1 flower. Palea-like organs with trichomes are formed in whorl 2 (arrowhead). Three ectopic palea-like organs (arrows) were removed to show the inside of the flower. (B) An organ of undefined identity. Long hairs are indicated by arrowheads. (C) Enlargement of the hairs (arrowheads) formed at the margin of the organ in B. (D) A vertical view of the flower. Bifurcated floral meristems (arrowheads), producing organ primordia (arrows) indeterminately. (E) A longitudinal section of an organ of unknown identity (arrowhead) showing no apparent differentiation of gametophytic tissues inside. (F-I) Scanning electron micrographs showing early development of spw1-1 dl-sup1 flowers. (F) A spw1-1 dl-sup1 flower producing six organ primordia (one primordium is covered by the lemma) in whorl 3 (arrowheads). (G) Six primordia in whorl 3 develop into structures that are not found in the wild type (arrows), and the floral meristem becomes enlarged in the lemma-palea direction (arrowhead). (H) The central floral meristem proliferates to form a number of apices (arrowheads). (I) Additional organs (arrowheads) are formed in an irregular arrangement from each meristematic apex. Bars: 500 µm (A,B,E), and 50 µm (C,D,F-I).

View larger version (125K): [in a new window]   Fig. 7. OsMADS45 expression in the wild type and spw1 dl-sup double mutant. (A) Wild-type vegetative shoot, (B) wild-type inflorescence shoots, (C) wild-type flower and (D) spw1-1 dl-sup1 flower (longitudinal sections). In wild type, the expression was detected in lodicules (arrowhead), stamens (st) and the carpel (ca), but not in vegetative or inflorescence shoots. In the mutant flower, the expression was detected in organs formed in whorl 2 and inner regions SAM, shoot apical meristem; le, lemma; pa, palea; st, stamen primordia. The numbers, 1 and 2, indicate the primary and secondary inflorescence meristems, respectively. Bar, 50 µm.

View larger version (50K): [in a new window]   Fig. 8. Structure of SPW1 gene and the molecular analysis of spw1 mutations. (A) Genomic structure of the SPW1 gene. The SPW1 gene consists of seven exons and six introns. The length of introns are 101, 204, 173, 448, 2168 and 124 bp, respectively. In the spw1-1 mutant, the last base of third intron of G is mutated to A. In spw1-2, the first base of the fifth intron of G is changed to A. (B) Deduced cDNA and protein sequence of SPW1. Arrowheads indicate the position of introns. The MADS box is indicated by the thick line and the K-box by the thin line. The solid box indicates the two amino acid residues deleted in spw1-1. The dashed box shows the exon that is deleted in spw1-2. The genomic sequence is available at the GenBank (accession no. AF424549). (C) RNA blot analysis of the SPW1 transcript in spw1 mutants. 1 µg of poly(A) RNA from spw1 mutants and wild-type was subjected to gel electrophoresis and hybridization with an SPW1-specific probe. 1 kb-long SPW1 transcript was detected in the wild type as well as mutants, but with a lesser amount in mutants. Unspliced RNA was not detected in mutants. (D) The same blot was hybridized with the probe for ubiquitin genes (accession number AC103891). (E) SPW1 RT-PCR analysis of spw1 mutants. The amplification of the region between exon 1 and 6 showed that the spw1-2 mutation resulted in the deletion of an approximately 100 bp-long sequence from SPW1 mRNA.

View larger version (129K): [in a new window]   Fig. 9. SPW1 expression in wild-type, dl-sup1 and spw1-1 flowers. (A-D, I-L) Wild-type, (E-H) dl-sup1 and (M-P) spw1-1 flowers. (A-H) Non-radioactive, and (I-P) radioactive in situ hybridization. (A) SPW1 RNA was detected in the floral meristem where incipient lodicule (arrowhead) and stamen primordia were formed. (B-D) SPW1 expression was detected in developing stamen and lodicule primordia but not in the carpel primordium (B,C) and (D) in the anthers (arrows) and filaments but not in the young ovary (ov) with developing stigma. (E) The expression pattern of SPW1 in dl-sup1 background was indistinguishable from the wild type at the stage when whorl 4 showed no sign of differentiation. (F,G,H) Ectopic expression of SPW1 was detected in the region where ectopic stamens are formed (arrowheads). Ectopic expression of SPW1 is not detected in the center of the floral meristem throughout flower development (arrows). (I,K,M,O) Bright-field, and (J,L,N,P) dark-field micrographs. (I,J) Floral meristem of a developing wild-type flower. (K,L) A young wild-type flower with developing floral organ primordia. (M,N) Floral meristem of a developing spw1-1 flower. (O,P) A young spw1-1 flower with developing floral organ primordia. le: lemma, pa: palea, lo: lodicule, st: stamen, ca: carpel, ov: ovule. The numbers 2, 3 and 4, indicate the position of whorls 2, 3 and 4, respectively. Bars. 30 µm.

View larger version (21K): [in a new window]   Fig. 10. A model of DL and SPW1 functions in floral organ specification. The model is also based on data that indicate the presence of class A and C gene(s) in rice (for a review, see Goto et al., 2001). (A) In the wild-type flower, DL acts in whorl 4 and specifies carpel identity (Ca) together with class C gene(s). SPW1 acts in whorl 2 and 3 as one of the B function genes and specifies stamen identity (St) in whorl 3 together with class C gene(s) and lodicule identity (Lo) in whorl 2 together with class A gene(s). (B) In dl mutants, SPW1 function expands to whorl 4, which results in the homeotic transformation of the carpel into stamens. (C) In spw1 mutants, stamens are transformed into carpels by the ectopic activity of DL in whorl 3. Whorl 2 organs are specified as palea-like (Pl) through the class A function alone. (D) In the spw1-1 dl-sup1 double mutant, organs with unknown identity (Id) are formed in whorls 3 and 4 without class B or DL functions. Organs in whorl 2 are palea-like, the same as those formed in spw1 mutants, specified by the class A function alone (see text). Pa, palea.