SLOW WALKER2, a NOC1/MAK21 Homologue,Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.Morphogenesis requires tightly coordinated coupling of cellular activities, such as cell growth, cell division, and differentiation. In past decades, significant progress on cell cycle control has been achieved mostly in single-celled organisms and cultured mammalian cells. The elucidation of the cyclin/cyclin-dependent kinase checkpoint control, for example, provides insight into molecular mechanisms on how and when cells divide. Mechanisms coupling cell growth to environmental and developmental signals have also been investigated. Ribosome biogenesis, a key for rapid cell growth, is coupled with nutrient availability and stress signals via the TOR signaling pathway (Warner et al., 2001; Wullischleger et al., 2006). However, questions such as how the cell senses intrinsic cellular homeostatic signals remain to be addressed. For example, how ribosome dynamics and translational activities are measured and coupled to cytokinesis and cell differentiation, especially in the context of development of multicellular organisms.Female gametogenesis in Arabidopsis (Arabidopsis thaliana) is a unique system to address such questions in multicellular organisms. During female gametogenesis, the haploid functional megaspore undergoes continuous cell growth and three cycles of consecutive nuclear division without cytokinesis, giving rise to a giant eight-nucleate, coenocytic cell: the embryo sac. The size of the embryo sac increases about 6-fold without cytokinesis until it reaches its maximum during gametogenesis in maize (Zea mays; Dow and Mascarenhas, 1991). The two polar nuclei migrate toward the micropylar half of the embryo sac and eventually fuse to give rise to a diploid nucleus of the central cell. As the polar nuclei migrate, cellularization takes place simultaneously to divide the coenocytic embryo sac into seven cells of four cell fates: three antipodal cells, two synergid cells, one egg cell, and one central cell (Drews et al., 1998; Grossniklaus and Schneitz, 1998; Yang and Sundaresan, 2000; Wilson and Yang, 2004). Obviously, its haploid nature and coupling of cell growth, division, and cell fates make the female gametophyte a nice system to investigate how these cellular activities are coordinated in development.The temporal and spatial control of cell growth, the mitotic division cycles, and cell fate specification during female gametogenesis have been the focus of sexual plant reproduction research. Recently, genetic studies have identified gametophytic mutations that start to shed light on the genetic and molecular control of these processes. Mutations in genes involved in diverse cellular functions, including ANDARTA (Howden et al., 1998), GAMETOPHYTIC FACTOR1 (GFA1; Christensen et al., 1997), HADAD (Moore et al., 1997), LETHAL OVULE2 (Sheridan and Huang, 1997), LYSOPHOSPHATIDYL ACYLTRANSFERASE (Kim et al., 2005), NOMEGA (Kwee and Sundaresan, 2003), PROLIFERA (Springer et al., 1995), SLOW WALKER1 (SWA1; Shi et al., 2005), SUCCINATE DEHYDROGENASE (Leon et al., 2007), and TISTRYA (Howden et al., 1998), all result in defective gametophytic cell divisions, implying that progression of the mitotic cycle is critical for the formation of a functional female gametophyte. Loss-of-function mutations in the Arabidopsis RETINOBLASTOMA-RELATED PROTEIN1, a key negative regulator controlling the G1/S transition of the cell cycle, result in uncontrolled nuclear proliferation and cell fates, giving rise to embryo sacs with supernumerary nuclei that are irregular in size and partially enclosed by cell wall-like structures (Ebel et al., 2004). Loss of functions in CYTOKININ INDEPENDENT1 (Hejatko et al., 2003), DIANA/AGAMOUS-LIKE61 (Bemer et al., 2008), AGAMOUS-LIKE80 (Portereiko et al., 2006a), and NUCLEAR FUSION DEFECTIVE1 (Portereiko et al., 2006b) affect polar nuclear fusion and central cell development.Accumulating data suggest a key role of the nucleolus in cell survival and proliferation (Cockell and Gasser, 1999; Shaw and Doonan, 2005). A number of nucleolar proteins have been discovered to be involved in linking cell proliferation control and ribosome biogenesis in yeast (Srivastava and Pollard, 1999; Du and Stillman, 2002; Jorgensen et al., 2002; Zhang et al., 2002; Bernstein et al., 2007). Mutations in genes involved in RNA processing, including SWA1 (Shi et al., 2005), GFA1/CLO1, and ATROPOS (ATO; Moll et al., 2008; Liu et al., 2009; Yagi et al., 2009), lead to slow progression of the division cycle during female gametogenesis. Intriguingly, mutation in LACHESIS (LIS), coding for a putative splicing factor, promotes egg cell fate in the synergid and the central cell at the expense of the synergid and central cell fate (Groß-Hardt et al., 2007), suggesting that LIS plays a pivotal role in suppressing the egg cell fate in the synergid and the central cell as well as the central cell fate in antipodal cells. Similarly, cell fate changes have also been observed in gfa1/clo1 and ato mutants (Moll et al., 2008). These data imply that RNA processing and ribosome biogenesis play a key role in coordinating cell cycle progression and cell fate. Here, we report the genetic and molecular characterization of a swa2 mutation that impairs cell growth and cell division in Arabidopsis. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 (MAK21) that is essential for ribosome biogenesis in yeast. We also show that SWA2 interacts physically with NOC2 homologues in yeast cells. Together, these data indicate that SWA2 is most likely involved in ribosome biogenesis and essential for cell cycle progression in female gametophyte development in Arabidopsis.