Gene and Protein Expression Profiles in Rice Gametes and Zygotes: A Cue for Understanding the Mechanisms of Gametic and Early Zygotic Development in Angiosperms

Abstract

In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is special-ized, fuses with the sperm cell, and converts into an active zygote for early embryo-genesis remains unclear. This lack of knowledge is partly attributable to the diffi culty of direct analyses of gametes and zygotes in angiosperms. Cell type-specifi c tran-scriptomes were obtained by microarray analyses for egg cells, sperm cells and zygotes isolated from rice fl owers, and up-or down-regurated genes in zygotes after fertilization were identifi ed as well as genes enriched in male and female gametes. In addition to transcriptome, proteins expressing in egg and sperm cells were glob-ally detected by highly sensitive liquid chromatography coupled with tandem mass spectroscopy technology, and proteins that are specifi cally or predominantly expressing in gametes were also identifi ed by comparison of protein expression profi les between gametes and somatic cells/pollen grains. Several rice or Arabidopsis lines with mutations in genes identifi ed by these proteome/transcriptome analyses showed clear phenotypic defects in seed set or seed development. These fi ndings sug-gest that the cell type-specifi c proteome/transcriptome data for gametes/zygotes are foundational information toward understanding the mechanisms of gametic and early zygotic development in angiosperms. In angiosperms, the sporophytic generation is initiated by double fertilization, resulting in the formation of seeds (reviewed in Raghavan 2003). In double fertilization, one sperm cell from the pollen grain fuses with the egg cell, and the resultant zygote devel-ops into an embryo that transmits genetic material from the parents to the next genera-tion. The central cell fuses with the second sperm cell to form a triploid primary endosperm cell, which develops into the endosperm that nourishes the developing embryo/seedling (Nawaschin 1898 ; Guignard 1899 ; Russell 1992). The conversion of the egg cell into the zygote is completed by two serial gametic processes: plasmogamy, the fusion of the plasma membrane between male and female gametes, and karyogamy, fusion of the nuclei of the male and female gametes in the fused gamete. Thereafter, the zygotic genome switches on within hours of fertilization for subsequent development of zygotes (Meyer and Scholten 2007 ; Zhao et al. 2011 ; Nodine and Bartel 2012). As for molecular players of plasmogamy, GENERATIVE CELL-SPECIFIC 1/ HAPLESS 2 (GCS1/HAP2) and EGG CELL 1 (EC1) have been identifi ed as putative fusiogens for male and female gametes, respectively (Mori et al. 2006 ; von Besser et al. 2006 ; Sprunck et al. 2012). GCS1/HAP2 was identifi ed as a key male mem-brane protein with a single transmembrane domain and a histidine-rich domain in the extracellular region. Recently, Sprunck et al. (2012) indicated that small cysteine-rich EC1 proteins accumulated in storage vesicles in the Arabidopsis egg cell are secreted via exocytosis upon sperm cell attachment to the egg cell, and that the secreted EC1 proteins function in redistribution of GCS1/HAP2 proteins to the sperm cell surface, resulting in successful gamete fusion. In addition to these two possible fusiogens, other players should be identifi ed to understand the mechanisms in plasmogamy. Karyogamy is accompanied by the congression of the male nucleus to the female nucleus and subsequent nuclear fusion. Yeast mating is the most intensively inves-tigated karyogamy event, and cytoskeleton-dependent nuclear congression and chaperone/ER-protein-dependent nuclear fusion have been well studied (Kurihara et al. 1994 ; Melloy et al. 2009 ; Tartakoff and Jaiswal 2009). However, in angio-sperms, the mechanism in karyogamy is poorly understood, except that Bip, a chap-erone in the lumen of endoplasmic reticulum, and NFD1, a component of the mitochondrial ribosome, function in polar nuclei fusion in Arabidopsis (Portereiko et al. 2006 ; Maruyama et al. 2010). In contrast to animals and lower plants, which have free-living gametes, angio-sperm fertilization and subsequent events, such as embryogenesis and endosperm development, occur in the embryo sac, which is deeply embedded in ovular tissue. Diffi culties in directly researching the biology of the embedded female gameto-phyte, zygote, and early embryo have impeded investigations into the molecular mechanisms of fertilization and embryogenesis. Therefore, such studies have been conducted predominantly through analyses of Arabidopsis mutants or transformants coupled with live-imaging (Berger 2011 ; Hamamura et al. 2012). Alternatively, direct analyses using isolated gametes or zygotes are possible because procedures for isolating viable gametes have been established, and an in vitro fertilization (IVF) T. Okamoto