The rice wall-associated receptor-like kinase gene OsDEES1 plays a role in female gametophyte development

Abstract

The wall-associated kinase (WAK) gene family is a unique subfamily of receptor-like kinases (RLKs) in plants. WAK-RLKs play roles in cell expansion, pathogen resistance, and metal tolerance in Arabidopsis (Arabidopsis thaliana). Rice (Oryza sativa) has far more WAK-RLK genes than Arabidopsis, but the functions of rice WAK-RLKs are poorly understood. In this study, we found that one rice WAK-RLK gene, DEFECT IN EARLY EMBRYO SAC1 (OsDEES1), is involved in the regulation of early embryo sac development. OsDEES1 silencing by RNA interference caused a high rate of female sterility. Crossing experiments showed that female reproductive organs lacking OsDEES1 carried a functional defect. A detailed investigation of the ovaries from OsDEES1 RNA interference plants indicated that the knockdown of OsDEES1 expression did not affect megasporogenesis but that it disturbed female gametophyte formation, resulting in a degenerated embryo sac and defective seed formation. OsDEES1 exhibited a tissue-specific expression pattern in flowers and seedlings. In the ovary, OsDEES1 was expressed in the megagametophyte region and surrounding nucellus cells in the ovule near the micropylar region. OsDEES1 was found to be a membrane-localized protein with a unique sequence compared with other WAK-RLKs. These data indicate that OsDEES1 plays a role in rice sexual reproduction by regulating female gametophyte development. This study offers new insight into the functions of the WAK-RLK family.

Figure 1.

Analysis of the sterile phenotype in the OsDEES1 RNAi transgenic rice plants. A, Five-month-old wild-type (WT) and OsDEES1 RNAi plants. B, Panicles of wild-type and OsDEES1 RNAi plants. C, Sterility analysis of T0 OsDEES1 RNAi plants generated in 2005, 2006, and 2007, respectively. C, Control plant transformed with the empty RNAi vector pTCK303; W, wild-type plant. D, OsDEES1 expression analysis in RNAi transgenic plants. E, The expression of OsDEES1 and its homolog OsDEES1-L4 in the RNAi plants. [See online article for color version of this figure.]

Figure 2.

Floral morphology and caryopsis development in the OsDEES1 RNAi and wild-type plants (WT). A, Flowers and caryopses from wild-type and RNAi plants at different stages of reproduction. B, Statistical analysis of the caryopsis abortive rate at the end of the filling stage. [See online article for color version of this figure.]

Figure 3.

Pollen viability, pollen germination, and pollen tube growth in the OsDEES1 RNAi and wild-type (WT) plants. A, Pollen vitality assay by I₂-KI and TTC staining. Arrows indicate inviable pollen grains. B, Percentage of inviable pollen grains in the OsDEES1 RNAi and wild-type plants. C, Pollen germinated in the stigma and pollen tube growth in the ovary. Arrows indicate pollen tubes that reached the micropylar region of the ovule. Con, Empty vector transgenic control plant; Emasculation, the stamens were removed before the anther burst in wild-type plants. D, Statistical analysis of pollen tube growth to the ovule micropyle in wild-type and OsDEES1 RNAi plants.

Figure 4.

Female gametophyte development in the wild-type and OsDEES1 RNAi plants. A, Semithin sections of female gametophytes at different developmental stages. Megasporogenesis was normal in the wild-type (a–d) and RNAi (e–h) plants. Shown are the megasporocyte (a and e; arrows indicate the megaspore mother cell), dyad (b and f), tetrad (c and g; arrows indicate the four megaspores), and functional megaspore (d and h) stages. Megagametogenesis is shown in the wild type from the functional megaspore stage (i) to various embryo sac developmental stages (j, early stage; k, middle stage; l, late stage) and in the OsDEES1 RNAi plants at the middle embryo sac stage (m, the arrow indicates an arrested functional megaspore cell) and in the late embryo sac stage (n, the arrow indicates a completely degenerated embryo; o, the arrow indicates a deformed embryo sac; p, the arrow indicates a small and empty embryo sac). B, Morphology of an embryo sac 1 d after pollination from wild-type and RNAi plants as observed by confocal microscopy. Left, a normal wild-type embryo sac with the multicellular globular embryo (EM) and free endosperm nuclei (FEN) indicated by arrows; middle and right, abnormal RNAi embryos showing a degenerated embryo sac (DES) and empty sac with no embryo formation (EES). C, Statistical analysis of abnormal embryo sac formation in the OsDEES1 RNAi and wild-type plants.

Figure 5.

OsDEES1 expression pattern in various plant tissues and immunolocalization in the ovary. A, OsDEES1 expression in different tissues as detected by GUS staining. GUS staining was observed in the coleoptile (a), node (b and c), leaf tongue (d), anther (e), style and lodicules (f) but not in the leaf blade (g), root (h), or caryopses (i). B, OsDEES1 expression in ovarian sections as shown by immunohistochemistry. Positive GUS immunolocalization (brown) was detected in the ovule near the micropyle (circumscribed region) and lodicules (long arrow) in OsDEES1_pro::GUS transgenic plants (b) but not in negative controls immunized with preimmune serum (a). In the enlarged view of the ovule (c), positive GUS immunolocalization was observed in the gametophyte region (white arrowhead) and surrounding ovule nucellus cells (white arrows). LO, Lodicule; OV, ovule; OW, ovary wall.

Figure 6.

Sequence analysis and subcellular localization of OsDEES1. A, Similarity analysis of OsDEES1 and AtWAKs using a protein sequence alignment (DNAMAN software). The signal peptide (SP), EGF2-like region (EGF2), calcium-binding EGF-like domain (EGF-Ca), transmembrane region (TM), ATP-binding region (ATP-B), and kinase active site (KAS) are underlined. Conserved amino acid residues in the EGF-like repeats are marked by asterisks. B, Phylogenetic tree analysis of OsDEES1 and AtWAKs. C, Subcellular localization of OsDEES1 lacking the kinase domain in tobacco and onion epidermal cells. YFP was ubiquitously localized in the cytoplasm, whereas OsDEES1ΔK-YFP was localized to the plasma membrane region in tobacco cells. In onion cells after plasmolysis, OsDEES1ΔK-YFP was detected in the plasma membrane region, in the cell wall (long arrow), and in thread-like structures (short arrow) between the cell wall and plasma membrane. The right-most images offer enlarged views of the circumscribed region.