Identification of genes expressed in the angiosperm female gametophyte

Gary N. Drews 1 Dongfang Wang 0 Joshua G. Steffen 1 Karen S. Schumaker 0 Ramin Yadegari 0 0 School of Plant Sciences, University of Arizona , Tucson, AZ 85721-0036 , USA 1 Department of Biology, University of Utah , Salt Lake City, UT 84112-0840 , USA Until recently, identification of gene regulatory networks controlling the development of the angiosperm female gametophyte has presented a significant challenge to the plant biology community. The angiosperm female gametophyte is fairly inaccessible because it is a highly reduced structure relative to the sporophyte and is embedded within multiple layers of the sporophytic tissue of the ovule. Moreover, although mutations affecting the female gametophyte can be readily isolated, their analysis can be difficult because most affect genes involved in basic cellular processes that are also required in the diploid sporophyte. In recent years, expression-based approaches in multiple species have begun to uncover gene sets expressed in specific female gametophyte cells as a means of identifying regulatory networks controlling cell differentiation in the female gametophyte. Here, recent efforts to identify and analyse gene expression programmes in the Arabidopsis female gametophyte are reviewed. The female gametophyte is essential for angiosperm reproduction The plant life cycle alternates between a haploid gametophyte generation and a diploid sporophyte generation. In plants, specialized diploid cells (mother cells) within sporophytes undergo meiosis and give rise to haploid spores. Spores undergo cell proliferation and differentiation to develop into multicellular haploid gametophytes. A major function of the gametophyte generation is to produce haploid gametes. Fusion of egg and sperm gives rise to the sporophyte, thereby completing the life cycle (Raven et al., 2005). In lower plants, gametophytes typically are the dominant and free-living generation. In contrast, angiosperms have dramatically reduced gametophytes that are composed of very few cells and are embedded within the sexual organs of the flower. Angiosperms have two gametophytes: the female gametophyte (also referred to as the embryo sac or megagametophyte) and the male gametophyte (also referred to as the pollen grain or microgametophyte). The male - gametophyte develops within the stamens anther and is composed of two sperm cells encased within a vegetative cell (McCormick, 2004; Singh et al., 2008; Borg and Twell, 2010). The female gametophyte develops within the ovule, which is found within the carpels of the flower. The angiosperm female gametophyte most commonly consists of one egg cell, one central cell, two synergid cells, and three antipodal cells (Drews and Yadegari, 2002; Colombo et al., 2008a; Sundaresan and Alandete-Saez, 2010; Yang et al., 2010). The female and male gametophytes are essential for the angiosperm reproductive process. Sexual reproduction is initiated when pollen is transferred from anther to stigma. Soon thereafter, the male gametophyte forms a pollen tube that grows through the carpels internal tissues and eventually releases its two sperm cells into one of the two synergid cells. Soon after arrival, the pollen tube ceases growth and the synergid cell penetrated by the pollen tube The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: undergoes cell death. Next, one sperm cell migrates to the egg cell and the other to the central cell. When the plasma membranes of the male gametes fuse with those of the egg and central cells, the sperm nuclei are transmitted into these cells for karyogamy. Following fertilization, the egg and central cell give rise to the seeds embryo and endosperm, respectively (Lord and Russell, 2002; Weterings and Russell, 2004; Berger et al., 2008; Sprunck, 2010). Female gametophyte cells control many steps of the fertilization process. During pollen tube growth, the synergid cells produce a guidance cue that directs pollen tube growth to the ovule and female gametophyte (Higashiyama and Hamamura, 2008; Okuda et al., 2009). Female gametophyte cells (mainly the synergid cells) contain factors that control arrest of pollen tube growth and release of pollen tube contents (Huck et al., 2003; Rotman et al., 2003, 2008; Escobar-Restrepo et al., 2007; Capron et al., 2008; BoissonDernier et al., 2009; Miyazaki et al., 2009; Tsukamoto et al., 2010). Upon fertilization, the ovule is induced to develop into a seed. Central cell-expressed gene products control the activation of endosperm development via epigenetic mechanisms (Kohler and Grossniklaus, 2005; Huh et al., 2008; Berger and Chaudhury, 2009; Jullien and Berger, 2009). Also, factors present in the egg cell may regulate the activation of embryo development, although these factors have yet to be identified (Curtis and Grossniklaus, 2008). Finally, genetic studies indicate that the female gametophyte plays a role in maternal control of embryo and endosperm development following fertilization (Evans and Kermicle, 2001; Grini et al., 2002). The mechanisms that control the differentiation of the female gametophyte and its individual cell types remain to be determined. However, recent work using molecular and genetic approaches has identified auxin as a critical signal for the proper development of the asymmetric structure of the female gametophyte in Arabidopsis (Pagnussat et al., 2009). Independent processes have also been shown to control cell identity particularly in the micropylar cells of the female gametophyte (Gross-Hardt et al., 2007; Pagnussat et al., 2007; Moll et al., 2008). Recent efforts in deciphering the regulatory networks that control female gametophyte cell differentiation and development are summarized below. Identification of genes expressed in the Arabidopsis female gametophyte As discussed above, each of the female gametophytes four cell types contains unique structural, developmental, and physiological features. Our goal is to understand the gene regulatory circuitry operating in these cells that causes them to acquire their unique features and functions during cell differentiation. As a first step toward dissecting these generegulatory networks, genes expressed in the Arabidopsis female gametophyte were identified using differential expression screens. The general strategy used to identify female gametophyteexpressed genes was to identify mRNAs present in normal ovules but not in mutant ovules lacking female gametophytes. male sterility1 (ms1) (Thorlby et al., 1997; Wilson et al., 2001; Ito et al., 2007) was used as the source of normal ovules, and determinant infertile1 (dif1) (Bai et al., 1999; Bhatt et al., 1999; Cai et al., 2003) as the source of mutant ovules lacking female gametophytes. ms1 is male sterile but undergoes normal ovule and female gametophyte development (Steffen et al., 2008). dif1 is a recessive sporophytic mu (...truncated)