The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa

Abstract

In this article, we report that carpel specification in the Oryza sativa (rice) flower is regulated by the floral homeotic gene DROOPING LEAF (DL) that is distinct from the well-known ABC genes. Severe loss-of-function mutations of DL cause complete homeotic transformation of carpels into stamens. Molecular cloning reveals that DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. DL is expressed in the presumptive region (carpel anlagen), where carpel primordia would initiate, and in carpel primordia. These results suggest that carpel specification is regulated by DL in rice flower development. Whereas CRC plays only a partial role in carpel identity, DL may have been recruited to have the more essential function of specifying carpels during the evolution of rice. We also show that DL interacts antagonistically with class B genes and controls floral meristem determinacy. In addition, severe and weak dl alleles fail to form a midrib in the leaf. The phenotypic analysis of dl mutants, together with analyses of the spatial expression patterns and ectopic expression of DL, demonstrate that DL regulates midrib formation by promoting cell proliferation in the central region of the rice leaf.

Figure 1.

Flower Phenotypes and Expression Patterns of OSH1. (A) Wild-type flower. (B) Complete transformation of carpels into stamens in the dl-sup1 flower. (C) Partial transformation of carpels in the intermediate dl-3 flower. (D) Multiple carpels produced in the dl-3 flower. (E) to (G) Spatial expression patterns of OSH1 in the wild-type flower. (H) Spatial expression patterns of OSH1 in dl-sup1. Arrowheads indicate ectopic stamens. ca, carpel; lo, lodicule; ov, ovule; st, stamen. Bars = 20 μm.

Figure 2.

Leaf Phenotypes. (A) and (B) Cross-section of the leaf blade of the wild type (A) and dl-sup1 (B). The bracket indicates the midrib structure. (C) to (E) Enlarged views of the inner structure at the adaxial sides of the wild-type midrib (C), indicated by the arrow in (A); the wild-type lateral vein (D), indicated by the arrowhead in (A); and the dl-sup1 central large vein (E), indicated by the arrow in (B). (F) and (G) Cross-section of the shoot apex of the wild type (F) and dl-sup1 (G). Arrowheads indicate the central vascular bundles, and asterisks indicate the central regions of the leaves that differ in the wild type and dl-sup1. asv, adaxial small vascular bundle; cc, clear cells; clv, central large vascular bundle. Bars = 100 μm.

Figure 3.

Gene Isolation and Structural Features of DL. (A) Cosegregation of the TOS17 insertion with DL genotypes. The genotypes, which were determined by the drooping leaf phenotype of the next generation (R₃), are shown above the lanes. The 5.5-kb XbaI band corresponds to the wild-type allele, whereas the 3.2-kb band is produced by the insertion of TOS17, which contains an internal XbaI site. (B) Genomic structure of DL and mutations in the six dl alleles. Boxes indicate exons and thick lines indicate introns. The coding regions are shown by shaded boxes. (C) Zinc-finger domain. The five conserved Cys residues are indicated with asterisks. (D) YABBY domain. The regions containing both domains have been extended slightly beyond the original definition (Bowman and Smyth, 1999) through the accumulation of more YABBY sequences. Amino acids identical to those of DL are indicated with black boxes. (E) Phylogeny of the YABBY gene family. The tree was constructed by the neighbor-joining method (Saitou and Nei, 1987). Numbers denote bootstrap values. Amino acid sequences of HvDL (Hordeum vulgare [barley] DL homolog) and LeDL (Lycopersicon esculentum [tomato] DL homolog) were deduced from the following EST sequences: HvDL (BG369278, AL509850) and LeDL (AI485831, AI483816). Rice FIL-like proteins have been described by Sawa et al. (1999).

Figure 4.

Scanning Electron Micrographs of Carpel Development and in Situ Localization of DL Transcripts. (A) Scanning electron micrograph of the wild-type floral meristem just before carpel initiation. (B) and (C) Scanning electron micrographs of carpel primordia and the flower meristem in the wild-type flower. (D) to (I) Localization of DL transcripts in the wild-type flower ([D] to [H]) longitudinal sections; [I] transverse section). Sectioned planes are indicated in (C) by a solid line (for [E] and [G]) and by a dashed line (for [D], [F], and [H]). (J) to (L) Localization of DL transcripts in the spw1 flower. The flower stage shown in (J) is the same as that shown in (D). Arrowheads in (A), (E), and (F) indicate the carpel anlagen. Arrows in (E) and (G) are DL signals in the region that correspond to the midrib in the lemma. Arrows in (J) to (L) indicate ectopic carpels in whorl 3. ca, carpel; epa, ectopic palea-like organ; fm, floral meristem; ov, ovule; st, stamen. Bars = 20 μm.

Figure 5.

Localization of DL Transcripts in Developing Wild-Type Leaves and an Embryo. (A) to (C) DL expression in leaves. Longitudinal (A) and transverse ([B] and [C]) section of a vegetative shoot apex. P1 and P2 leaf primordia and the shoot apical meristem are outlined in (B). The section of (C) was taken 100 μm above that shown in (B). (D) DL expression in an embryo 10 d after pollination, when two leaves had initiated. Plastochron numbers are indicated as P1 to P5. Asterisks indicate the shoot apical meristem. The arrow shows the abaxial mesophyll. co, coleoptyle. Bars = 20 μm.

Figure 6.

Phenotypes of ACTIN:DL Plants. (A) and (D) Morphology of leaf blades. (B) and (E) Transverse sections of the leaf blades of seedlings. (C) and (F) Transverse sections of developing (P3 to P4) leaf blades. (G) and (H) Enlarged view of the regions shown by the brackets in (B) and (E), respectively. (A), (B), (C), and (G) Wild-type. (D), (E), (F), and (H) ACTIN:DL. Arrowheads indicate adaxial small vascular bundles. ab, abaxial; ad, adaxial; asv, adaxial small vascular bundle; bc, bulliform cells; p, phloem; sc, sclerenchyma; x, xylem. Bars = 200 μm.