Female Gametophyte Development

Early in their evolution, plants acquired a life cycle that alternates between a multicellular haploid organism, the gametophyte, and a multicellular diploid organism, the sporophyte. Angiosperms have both female and male gametophytes. The female gametophyte is critical to many steps of the angiosperm reproductive process, including pollen tube guidance, fertilization, the induction of seed development upon fertilization, and maternal control of seed development after fertilization. Genetic analysis in Arabidopsis and maize has revealed mutants defective in almost all stages of female gametophyte development, and analysis of these mutants is beginning to reveal features of the female gametophyte developmental program. In addition, mutants defective in female gametophyte function have revealed regulatory genes required for the induction of endosperm development. From these studies, we are beginning to understand the regulatory networks involved in female gametophyte development and function. Gametophytes and sporophytes differ morphologically and functionally. The major function of diploid sporophyte generation is to produce haploid spores, which are the products of meiosis. Spores undergo cell proliferation and differentiation to develop into gametophytes. The major function of gametophyte generation is to produce haploid gametes. The fusion of egg and sperm gives rise to the zygote, which is the beginning of diploid sporophyte generation, thereby completing the life cycle (Gifford and Foster, 1989). The Arabidopsis Female Gametophyte. (A) Ovule. (B) Female gametophyte. The view in (B) is perpendicular to that in (A). The mature female gametophyte in Arabidopsis is ∼105 μm long and ∼25 μm wide. The gray areas represent cytoplasm, the white areas represent vacuoles, and the black areas represent nuclei. ac, antipodal cells; cc, central cell; ch, chalazal region of the ovule; ec, egg cell; f, funiculus; mp, micropyle; sc, synergid cell; sn, secondary nucleus. The angiosperm gametophytes are essential for the reproductive process. During sexual reproduction in angiosperms, the male gametophyte is transferred from the anther to the carpel's stigma, whereupon it forms a pollen tube that grows great distances through the carpel's internal tissues to deliver its two sperm cells to the female gametophyte. One sperm cell fertilizes the egg cell and the second sperm cell fuses with the central cell. After fertilization, the ovule gives rise to a seed; the seed's embryo, endosperm, and seed coat are derived from the fertilized egg cell, fertilized central cell, and ovule integuments, respectively (Maheshwari, 1950). The female gametophyte plays a critical role in essentially every step of the reproductive process. During pollen tube growth, the female gametophyte participates in directing the pollen tube to the ovule (Higashiyama, 2002; Johnson and Preuss, 2002; Higashiyama et al., 2003). During fertilization, cytoskeletal components within the female gametophyte direct the sperm cells to the egg cell and the central cell (Russell, 1992, 1993; Lord and Russell, 2002). Upon fertilization, female gametophyte–expressed genes control the initiation of seed development (Chaudhury et al., 2001). During seed development, female gametophyte–expressed gene products play a role in controlling embryo and endosperm development (Ray, 1997; Chaudhury and Berger, 2001). In this review, we describe angiosperm female gametophyte structure and development, summarize the female gametophyte's reproductive functions, and discuss the molecular and genetic approaches that are being used to understand these processes at the molecular level. Comprehensive reviews of the female gametophyte have been published previously (Maheshwari, 1950; Willemse and van Went, 1984; Haig, 1990; Huang and Russell, 1992; Russell, 2001). Patterns of Female Gametophyte Development Exhibited by Angiosperms. Genera exhibiting these patterns are indicated in parentheses. More comprehensive descriptions of the variation among angiosperms can be found in several reviews (Maheshwari, 1950; Willemse and van Went, 1984; Haig, 1990; Huang and Russell, 1992; Russell, 2001). In this figure, the chalazal end of the female gametophyte is up and the micropylar end is down. FG, female gametophyte. During megasporogenesis, the diploid megaspore mother cell undergoes meiosis and gives rise to four haploid nuclei. Angiosperms exhibit three main patterns of megasporogenesis, referred to as monosporic, bisporic, and tetrasporic. These three patterns are summarized in Figure 2. The three types differ mainly in whether cell plate formation occurs after these divisions, thus determining the number of meiotic products that contribute to the formation of the mature female gametophyte. In the monosporic pattern, both meiotic divisions are accompanied by cell plate formation, resulting in four one-nucleate megaspores. Subsequently, three megaspores, generally the micropylar-most megaspores, undergo cell death. In the bisporic pattern, cell plates form after meiosis I but not meiosis II. The result is two two-nucleate megaspores, one of which degenerates. In the tetrasporic pattern, cell plates fail to form after both meiotic divisions, resulting in one four-nucleate megaspore. Thus, these three patterns give rise to a single functional megaspore that contains one (monosporic), two (bisporic), or four (tetrasporic) meiotic nuclei. The monosporic pattern is the most common form and is represented within the Polygonum pattern (Maheshwari, 1950; Willemse and van Went, 1984; Haig, 1990; Huang and Russell, 1992). Female Gametophyte Development in Arabidopsis. The steps are described in the text and by Christensen et al. (1997). Category designations show the developmental stage affected in the female gametophyte mutants. Phenotypic categories are as follows: category 1, megaspores fail to undergo cell death; category 2, megaspores do not progress beyond stage FG1; category 3, pleiotropic defects during the nuclear division phase; category 4, failure to cellularize or abnormal cell shape; category 5, polar nuclei fail to fuse; category 6, antipodal cells fail to undergo cell death; category 7 (not shown), morphologically wild-type female gametophytes at the terminal stage. The gray areas represent cytoplasm, the white areas represent vacuoles, and the black areas represent nuclei. In this figure, the chalazal end of the female gametophyte is up and the micropylar end is down. ac, antipodal cells; cc, central cell; ec, egg cell; fm, functional megaspore; m, megaspore; mmc, megaspore mother cell; pn, polar nuclei; sc, synergid cell; sn, secondary nucleus. Throughout development, the female gametophyte exhibits a polarity along its chalazal-micropylar axis. During Polygonum-type megasporogenesis, the chalazal-most megaspore survives and the other three megaspores undergo cell death (Figure 3). During cell differentiation, the nuclei at the micropylar end become specified to develop into the egg cell, the micropylar polar nucleus, and the synergid cells; the chalazal nuclei develop into the three antipodal cells and the chalazal polar nucleus (Figure 3). Furthermore, all of the cells within the female gametophyte differentiate into polar structures. For example, in many species, the egg cell's nucleus is located toward the chalazal end and its vacuole occupies the micropylar end; by contrast, the synergid and central cells have the opposite polarity (Figure 3) (Willemse and van Went, 1984; Huang and Russell, 1992; Christensen et al., 1997). Thus, the establishment of polarity within the female gametophyte corresponds to the asymmetric development of the surrounding ovule layers, suggesting that female gametophyte polarity is regulated, at least in part, by the surrounding sporophytic tissues. Sporophytic factors that influence female gametophyte development have yet to be identified. Soon after pollen is transferred from anther to stigma, the male gametophyte forms a pollen tube that grows via a tip-growth process through the carpel's sporophytic tissue to reach the female gametophyte. The pollen tube enters the female gametophyte by growing into one of the two synergid cells through a structurally elaborated portion of the micropylar cell wall known as the filiform apparatus. The penetrated synergid cell undergoes cell death soon before or upon pollen tube arrival. Immediately after arrival, pollen tube growth ceases, an aperture forms at or near the pollen tube tip, and the contents of the pollen tube, including the two sperm cells, are released rapidly into the degenerating synergid cytoplasm. Double fertilization occurs when the two sperm cells migrate to the egg and central cells and their plasma membranes fuse with the respective target cell to transport the sperm nuclei for karyogamy (van Went and Willemse, 1984; Russell, 1992, 1996). Because of their two-staged life cycle, plants possess two broad classes of mutations: sporophytic mutations and gametophytic mutations. Sporophytic mutations affect the diploid sporophyte phase of the plant life cycle and exhibit Mendelian 1:2:1 segregation patterns. Gametophytic mutations, by contrast, affect the haploid gametophyte phase of the plant life cycle and are not transmitted through egg and/or sperm. As a consequence, gametophytic mutations exhibit non-Mendelian segregation patterns and can only be transmitted from generation to generation as heterozygotes. For example, in a self cross of a heterozygous individual (e.g., genotype A/a), female gametophyte–specific mutations (i.e., mutations that affect the female gametophyte but not the male gametophyte) segregate 1:1 for A/A:A/a progeny (Moore et al., 1997; Drews et al., 1998; Drews and Yadegari, 2002; Page and Grossniklaus, 2002). Sporophytic and gametophytic mutations affect different aspects of female gametophyte development. Sporophytic mutations affect those aspects that occur during the diploid phase, including megaspore mother cell development, meiosis, and control of female gametophyte development by the surrounding sporophytic tissue (e.g., female gametophyte polarity; discussed above). Sporophytic mutations that affect these processes are identified in screens for female-sterile mutants (Chaudhury et al., 1998; Gasser et al., 1998; Grossniklaus and Schneitz, 1998; Schneitz et al., 1998; Schneitz, 1999). Gametophytic mutations affect those aspects of female gametophyte development that occur after meiosis, including megagametogenesis and functioning of the mature female gametophyte (pollen tube guidance, fertilization, induction of seed development, or maternal control of seed development). Gametophytic mutants typically are identified using two criteria: reduced seed set and segregation distortion. Reduced seed set results because on a plant heterozygous for a female gametophyte mutation, approximately half of the female gametophytes are mutant and nonfunctional; thus, they fail to undergo normal seed development. Segregation distortion results because, as described above, gametophytic mutations are transmitted to subsequent generations at reduced frequency (Moore et al., 1997; Drews et al., 1998; Drews and Yadegari, 2002; Page and Grossniklaus, 2002). As discussed above, the sporophytic tissue surrounding the female gametophyte may play a role in controlling megagametogenesis. Consistent with this notion, megagametogenesis is affected in most sporophytic ovule-development mutants (Chaudhury et al., 1998; Gasser et al., 1998; Grossniklaus and Schneitz, 1998; Schneitz et al., 1998; Schneitz, 1999). Mutants defective specifically in the sporophytic control of megagametogenesis should exhibit defects in megagametogenesis but not in the sporophytic parts of the ovule. Mutants with this phenotype have been reported (Schneitz et al., 1997). However, the genes affected in these mutants have not been identified; thus, the molecular basis of these sporophytic effects remains to be determined. During the last few years, many gametophytic mutants affected in female gametophyte development have been identified and analyzed (Christensen et al., 2002; Drews and Yadegari, 2002; Grini et al., 2002; Huck et al., 2003; Rotman et al., 2003). We have identified and analyzed >150 mutants, allowing genetic dissection of the female gametophyte developmental pathway (Christensen et al., 2002; D. Otsuga, C. Dever, N. Huefner, L.A. Ogden, L.G. Jones, and G.N. Drews, unpublished data). Most of these mutants are affected in megagametogenesis. As summarized in Figure 3, the megagametogenesis mutants fall into six phenotypic categories corresponding to key developmental events involved in the formation of a mature female gametophyte. Although the genes affected for most of the megagametogenesis mutants are not known yet, the use of insertional mutagens (e.g., T-DNA or transposons) should facilitate their rapid identification. Certain key features of megagametogenesis mutant screens will enable a comprehensive analysis of gametophyte generation and also will contribute to an understanding of important cellular and developmental processes throughout the plant life cycle. First, these screens have yielded mutants with defects throughout megagametogenesis. Based on these phenotypes, the affected genes can be ordered, to some extent, within the developmental program. In addition, many mutations exhibit defects in specific cellular processes, including mitosis, vacuole formation, cell wall formation, nuclear fusion, and cell death. Significantly, many of these mutations would not be identified in typical sporophytic screens because they cannot become homozygous during sporophyte generation. Therefore, genetic analysis of these key processes likely will rely on the characterization of defective gametophytes. During the final stages of pollen tube growth, the pollen tube grows toward an ovule and then up the surface of the funiculus until it enters the micropyle to penetrate the female gametophyte (Higashiyama, 2002; Johnson and Preuss, 2002; Higashiyama et al., 2003). This directed growth pattern suggests that the ovule and female gametophyte play a role in guiding pollen tube growth. Many studies suggest that pollen tube guidance is controlled by both sporophytically expressed and expressed factors and Preuss, 2002; 2002; Johnson and Preuss, 2002; Higashiyama et al., 2003). identified as a sporophytic that pollen tube growth and guidance et al., 2003). whether the female gametophyte plays a role in pollen tube guidance, several analyzed pollen tube growth patterns in Arabidopsis mutants defective in embryo development. studies have that female gametophytes fail to pollen suggesting that the embryo is the of an that the pollen tube to the ovule et al., et al., 1997; et al., and of pollen tube growth patterns in Arabidopsis mutants defective in female gametophyte development suggests that guidance by the female gametophyte In mutants in which female gametophyte development is affected (e.g., mutants in categories to in Figure pollen fail to along the funiculus et al., et al., 1997). contrast, in mutants in which female gametophyte development is affected (e.g., mutants, which exhibit development and have polar nuclei at the of pollen along the funiculus but do not the micropyle and These suggest that guidance by the female gametophyte at least two guidance from the to the funiculus guidance and guidance from the funiculus to the micropyle guidance and the specific cells within the female gametophyte that are the of the pollen tube Higashiyama and in an in pollen using from In this species, of the embryo from the ovule and is to cells and in which the egg cell and central cell pollen with synergid cells to pollen et al., 2001). These studies the synergid cells as the of the pollen tube in the in also that the of pollen tube is to μm and that the is specific et al., 2003; 2002). is the of the synergid cell guidance has been to be a guidance because it can pollen in in some and et al., is in in synergid cells and 1993; and Russell, and is for pollen tube growth et al., et al., 1997; et al., 1999). However, the of to the in the in not affect pollen tube that is not a (Higashiyama, 2002; Higashiyama et al., 2003). The in pollen tube guidance facilitate the of the guidance an of this is likely to be the on the of the target the synergid screens for female gametophyte mutants with defects specifically in pollen tube guidance may facilitate the of the Female gametophyte mutants defective specifically in pollen tube guidance have yet to be Thus, the of the synergid cell guidance remains to be determined. The pollen tube enters the female gametophyte by growing into one of the synergid Soon the pollen tube growth, at or near its tip, and its The synergid cell penetrated by the pollen tube undergoes cell before or upon pollen tube thus, the pollen tube contents are released into the degenerating of the synergid cell (van Went and Willemse, 1984; Willemse and van Went, 1984; Russell, 1992, 2002; Lord and Russell, 2002; 2003). The of these events has been in Arabidopsis et al., 2002). cell death may be a for normal fertilization in For example, the process of to both pollen tube and sperm cell during fertilization (Willemse and van Went, 1984; Huang and Russell, 1992). In addition, synergid generally is accompanied by cytoskeletal that may facilitate male from the pollen tube to the egg and the central cell (Russell, 1993; et al., In some species, synergid cell death to be a final step of the megagametogenesis developmental program. contrast, in other species, including synergid is not an of the megagametogenesis process because synergid cell death not occur is (van Went and Willemse, 1984; Willemse and van Went, 1984; Russell, 1992; Christensen et al., 1997; 2001). and not be for synergid cell death in as Arabidopsis. First, synergid may be a process (e.g., the of pollen tube contents into the synergid cell may the synergid synergid cell death may be a process by synergid cell death via a the pollen or from female or through direct with the synergid cell (van Went and Willemse, 1984; Willemse and van Went, 1984; Russell, 1992; 2002). of these may in For example, in some species, the synergid cell to be at the of pollen tube suggesting via a process. contrast, in other species, synergid cell death to be before pollen tube at the female gametophyte, suggesting that a synergid cell death (van Went and Willemse, 1984; Willemse and van Went, 1984; Russell, 1992; 2002). In the of synergid cell death at the of pollen tube at the female gametophyte has not been determined. However, as discussed analysis of several female gametophyte mutants suggests that synergid cell death in Arabidopsis is an process. Because fertilization within the embryo it is likely that many female gametophyte–expressed gene products are for this process. However, because the of fertilization are a molecular understanding of the angiosperm fertilization process has been The development of in fertilization et al., and and should the and dissection of fertilization Lord and Russell, 2002). For example, the in fertilization facilitate the of all including and within of gametophytes and after fertilization. is the of mutants defective in the fertilization process. During the last years, several female gametophyte mutants affected in the fertilization process have been including gametophytic (Christensen et al., et al., and et al., 2003). In all three mutants, embryo development is normal and or essentially normal female gametophytes also have defects in fusion of the polar and mutant female gametophytes pollen but fail to become In to embryo undergo synergid cell death et al., by contrast, and embryo fail to undergo synergid cell death after (Christensen et al., 2002; Rotman et al., 2003). The and mutations do not affect megaspore or antipodal cell suggesting that synergid cell death has The mutant has an also by the wild-type pollen mutant female gametophytes but fail to growth, and their contents et al., 2003; Rotman et al., 2003). it is whether the synergid cell death is a secondary of the pollen tube or the of these two mutants that they are defective in some of a that in the and the is required for function. These suggest that synergid cell death functional which also are required for cell death in (Christensen et al., 2002). aspects of the synergid cell death process to be determined. The by and are of the and mutants several aspects of the fertilization process. First, and do not affect pollen tube and should not the of the synergid cell, suggesting that synergid cell death in Arabidopsis is not a process. and embryo fail to undergo synergid cell death and yet pollen suggesting that synergid cell death is not required for pollen tube the abnormal pollen tube within and female gametophytes suggests that the of synergid cells not normal pollen tube and within the female gametophyte (Christensen et al., 2002; Huck et al., 2003; Rotman et al., 2003). genetic that the female gametophyte seed development at several First, the female gametophyte the initiation of seed development by a set of that this process in the of fertilization. the female gametophyte contains factors before fertilization that are required for embryo and endosperm development after fertilization. the female gametophyte plays a role in controlling the of genes required for seed development. Female gametophyte mutations that affect the initiation of seed development endosperm et al., Chaudhury et al., (Chaudhury et al., 1997; Grossniklaus et al., and (Chaudhury et al., 1997). In all three mutants, endosperm development occurs in the of fertilization. The and are to involved in the of gene in and et al., 1998; et al., et al., et al., 1999). The and genes are expressed in the female gametophyte, in the central cell, before fertilization et al., et al., et al., et al., these suggest that the female gametophyte a set of that endosperm development before fertilization et al., this fertilization to the initiation of endosperm development by the et al., 1999). fertilization this remains an The most likely endosperm development by the of target genes involved in this process. One target identified et al., 2003). a have that and with the In the occurs in the stages of endosperm development and is not in the female gametophyte before fertilization. also is expressed in suggesting a function in both embryo and However, is in and and is in female gametophytes. After fertilization, exhibit endosperm and embryo et al., 1998; et al., 1999). These defects are in part, to the of because reduced of the seed phenotype et al., 2003). After the initiation the female gametophyte seed development by maternal required for this the female gametophyte contains maternal factors before fertilization that are required for embryo and endosperm development after fertilization. Female gametophyte–expressed genes required for embryo and endosperm development are referred to as gametophytic genes (Ray, 1997; Drews et al., 1998; Drews and Yadegari, 2002). This is in to sporophytic genes (e.g., Arabidopsis and and which are expressed in sporophytic tissue et al., et al., et al., 1997). The of gametophytic mutants described to the Arabidopsis and mutants et al., the Arabidopsis mutant et al., and the maize maternal mutant and 2001). the of which at the four-nucleate stage of development et al., female gametophytes in these mutants the development of the embryo or endosperm or both is affected during seed development. For example, from female gametophytes exhibit defects as as the stage and fail to progress beyond the stage et al., 2002). The and genes have not been thus, the molecular basis for the gametophytic maternal effects in these mutants remains to be determined. However, a of the in the female gametophyte to be required for normal embryo development et al., at which the female gametophyte seed development is through the control of important of seed development is the of genes required during this process. is by the from the male or female gametophyte. For example, with and the maternal are but the are during endosperm development et al., et al., The is via of the maternal or a process generally with the of within and the region of the target gene and 2003). important is when the are during development. the is that the are during the male gametophyte is that both become at some during the life cycle and the are during the female gametophyte The to be the with the suggests that of the maternal of are in the female gametophyte's central cell by a is a to the of et al., 2002). is expressed in the female gametophyte's central cell before fertilization. is reduced in female gametophytes and the endosperm of derived from embryo likely the gene by structure through the of from its and in the female gametophyte. In the female gametophyte the maternal of and other regulatory genes for after fertilization. The function of in gene may not be because mutations in a have been to of a and a gene et al., 2002). The female gametophyte an essential portion of the plant life cycle several reproductive processes, including pollen tube guidance, fertilization, the induction of seed development, and maternal control of seed development. During the few years, genetic approaches in Arabidopsis and maize have with molecular and genetic megagametogenesis and the induction of seed development. However, we pollen tube guidance, fertilization, and the maternal control of seed development at the molecular level. In addition, a comprehensive of the genes expressed in the female gametophyte has not been In of the that a of female gametophytic genes are to have or and Yadegari, a of and approaches will be required to understand the development and reproductive of the female gametophyte. We Johnson for critical of the on female gametophyte development by from the and to and from the of to