YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

Abstract

Background: In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.

Results: Here we report the characterization of a mutation, yaozhe (yao), that causes zygote arrest and misplacement of cell plate of the zygote, leading to early embryo lethality. In addition, gametophyte development is partially impaired. A small portion of the mutant embryo sacs are arrested at four-nucleate stage with aberrant nuclear positioning. Furthermore, the competence of male gametophytes is also compromised. YAO encodes a nucleolar protein with seven WD-repeats. Its homologues in human and yeast have been shown to be components of the U3 snoRNP complex and function in 18S rRNA processing. YAO is expressed ubiquitously, with high level of expression in tissues under active cell divisions, including embryo sacs, pollen, embryos, endosperms and root tips.

Conclusions: Phenotypic analysis indicated that YAO is required for the correct positioning of the first zygotic division plane and plays a critical role in gametogenesis in Arabidopsis. Since YAO is a nucleolar protein and its counterparts in yeast and human are components of the U3 snoRNP complex, we therefore postulate that YAO is most likely involved in rRNA processing in plants as well.

Figure 1

Phenotype of the yao plant. A-B: Photographs showing full seed set in wild-type (A) and partial seed set in yao/+ mutant siliques (B). C-L: Micrographs of cleared ovules from the same yao/+ silique showing the wild-type (+/+) embryo at the globular stage (C), and yao/yao mutant ovules (D-L). Mutant zygotes were either arrested (D-E) or divided once with misplaced division plane (F-G) and an enlarged apical cell (H-J). The abnormal apical cells divided transversely (K-L). ac, apical cell; bc, basal cell; ep, embryo proper; su, suspensor; z, zygote. Arrowheads indicating enlarged nucleoli. Bar = 20 μm.

Figure 2

Female gametophyte development in yao/+ siliques. Confocal micrographs showing a female gametophyte at four-nucleate stage (FG4) (A) and FG5 stages (B) from wild-type plants, female gametophytes (C-E) from the same yao/+ pistil arrested at FG4 with misplaced nuclei at the micropylar pole (C, D) and reached to the FG5 stage (E). Compare the positioning of nuclei in A and C-D. PN, polar nucleus; EN, egg nucleus; SN, synergid nucleus; V, vacuole. Arrows indicating nucleus of the female gametophyte. Bar = 20 μm.

Figure 3

Molecular characterization of the YAO. (A) Diagram of the Ds insertion in YAO. The grey boxes indicate the exons of YAO, the white boxes indicate the UTR regions and the lines indicate the intron regions. The nucleotide numbers are consistent with those in the Arabidopsis genome on TAIR web site. (B) Diagram of predicated domain structure of YAO protein. NLS, nuclear localization signal. (C) Phylogenetic tree of YAO with its homologs from other organisms: EMB2271 from A. thaliana; Os03G0625900 from O. sativa; HsU3-55K from H. sapiens; MmNP_663595.1 from M. musculus; XlAAI57488.1 from X. laevis; CeNP_872030.2 from C. elegans; SpNP_594143.1 from S. pombe; ScRrp9, from S. cerevisiae. (D) Protein alignment of YAO and EMB2271. The seven predicted WD40-repeat domains are underlined.

Figure 4

YAO is localized in the nucleolus. (A) Confocal micropgraphs showing nuclear localization of YAO-GFP in root tip. (B-D) Confocal micrographs of the same cell showing the subcellular localization of YAO-GFP (B), nucleolar marker SWA2-DsRed (C), and the merged image (D), note the nucleolar localization of YAO. (E-H) Confocal micrographs showing YAO-GFP localization in megaspore mother cell (E), embryo sac and integument cells (F), zygote and endosperm (G), endosperm and preglobular embryo (H) nuclei. ac, apical cell; ap, antipodal cell; bc, basal cell. e, endosperm; ec, egg cell; ms, megaspore mother cell; pn, polar nucleus; sg, synergid; z, zygote. Bar = 20 μm.

Figure 5

Expression pattern of YAO. GUS staining showing YAO expression in a 10-day-old seedling (A), root tip (B), and lateral root primordial (C), pollen grains (D), embryo sacs before fertilization (E), and embryo and endosperm (F). RNA in situ showing YAO mRNA in early embryo (G) and endosperm (H), and sense control (I). Bar: 1 mm (A), 20 μm (E-I), 50 μm (C, D), 100 μm (B).