Perspective: the origin of flowering plants and their reproductive biology--a tale of two phylogenies

Recently, two areas of plant phylogeny have developed in ways that could not have been anticipated, even a few years ago. Among extant seed plants, new phylogenetic hypotheses suggest that Gnetales, a group of nonflowering seed plants widely hypothesized to be the closest extant relatives of angiosperms, may be less closely related to angiosperms than was believed. In addition, recent phylogenetic analyses of angiosperms have, for the first time, clearly identified the earliest lineages of flowering plants: Amborella, Nymphaeales, and a clade that includes Illiciales/ Trimeniaceae/Austrobaileyaceae. Together, the new seed plant and angiosperm phylogenetic hypotheses have major implications for interpretation of homology and character evolution associated with the origin and early history of flowering plants. As an example of the complex and often unpredictable interplay of phylogenetic and comparative biology, we analyze the evolution of double fertilization, a process that forms a diploid embryo and a triploid endosperm, the embryo-nourishing tissue unique to flowering plants. We demonstrate how the new phylogenetic hypotheses for seed plants and angiosperms can significantly alter previous interpretations of evolutionary homology and firmly entrenched assumptions about what is synapomorphic of flowering plants. In the case of endosperm, a solution to the century-old question of its potential homology with an embryo or a female gametophyte (the haploid egg-producing generation within the life cycle of a seed plant) remains complex and elusive. Too little is known of the comparative reproductive biology of extant nonflowering seed plants (Gnetales, conifers, cycads, and Ginkgo) to analyze definitively the potential homology of endosperm with antecedent structures. Remarkably, the new angiosperm phylogenies reveal that a second fertilization event to yield a biparental endosperm, long assumed to be an important synapomorphy of flowering plants, cannot be conclusively resolved as ancestral for flowering plants. Although substantive progress has been made in the analysis of phylogenetic relationships of seed plants and angiosperms, these efforts have not been matched by comparable levels of activity in comparative biology. The consequence of inadequate comparative biological information in an age of phylogenetic biology is a severe limitation on the potential to reconstruct key evolutionary historical events.     

The evolution of double fertilization and endosperm: an ”historical” perspective

 One hundred years ago, the developmental origin of endosperm from double fertilization was discovered independently by Navashin and Guignard. For much of the twentieth century, specific events related to the evolutionary origin of the endosperm of flowering plants remained a mystery. However, during the past 20 years, advances in phylogenetic reconstruction of seed plants, genetic theory associated with kin selection, and comparative study of the reproductive biology of the closest living relatives of angiosperms (Gnetales) have advanced our understanding of the evolutionary events associated with the origin of double fertilization and endosperm. Recent developmental analyses of Ephedra and Gnetum (members of Gnetales) indicate that these nonflowering seed plants undergo a regular process of double fertilization that yields two diploid zygotes. Use of explicit genetic and developmental criteria for analysis of evolutionary homology demonstrates congruence with the hypothesis that double fertilization processes in Gnetales and angiosperms were inherited from a common ancestor of the two lineages. In its rudimentary form, the second fertilization event in the ancestors of flowering plants yielded a supernumerary diploid embryo that was genetically identical to the normal embryo, a process most similar to what occurs in extant Ephedra. Subsequent to the divergence of the angiosperm stem lineage, the supernumerary embryo derived from double fertilization was developmentally modified into an embryo-nourishing structure, endosperm, that now characterizes angiosperms. Received: 25 September 1997 / Accepted: 3 November 1997     

The evolution of embryogeny in seed plants and the developmental origin and early history of endosperm

For almost a century, the formation of endosperm from a second and distinctive fertilization event has been viewed as a unique feature of flowering plants. However, until recently, the evolutionary origin of this unique embryo-nourishing entity remained a mystery. Based upon comparative developmental analysis of reproduction among basal angiosperms and their closest extant relatives, the Gnetales (Ephedra, Gnetum, and Welwitschia), it is possible to construct an explicit hypothesis of the events that led to the evolutionary establishment of double fertilization and endosperm. The formulation of this historical record is derived entirely from and dependent upon the determination of reproductive features that are likely to have characterized the common ancestors of angiosperms and Gnetales. Current evidence is most congruent with the concept that a process of double fertilization first evolved in a common ancestor of the Gnetales and angiosperms. Initially, however, the second fertilization product was diploid and yielded a supernumerary embryo. Subsequent to the divergence of the angiosperm lineage from its closest relatives (which include the Gnetales), modification of the development of the supernumerary embryo (derived from the second fertilization event) led to the establishment of an embryo-nourishing endosperm. Comparative analysis of patterns of embryogeny within Gnetales and angiosperms establishes that embryo development in the ancestors of flowering plants (with a rudimentary process of double fertilization) was ab initio cellular, and not free nuclear, as had previously been assumed. Thus, it is likely that the earliest flowering plants displayed an ab initio cellular pattern of endosperm development, whose expression was inherited from that of the supernumerary embryo of the ancestors of flowering plants.     

植物胚乳发育的表观遗传学调控

被子植物的种子发育从双受精开始, 产生二倍体的胚和三倍体的胚乳。在种子发育和萌发过程中, 胚乳向胚组织提供营养物质, 因此胚乳对胚和种子的正常生长发育至关重要。开花植物发生基因组印迹的主要器官是胚乳。印迹基因的表达受表观遗传学机制的调控, 包括DNA甲基化和组蛋白H3K27甲基化修饰以及依赖于PolIV的siRNAs (p4-siRNAs)调控。基因组印迹的表观遗传学调控对胚乳的正常发育和种子育性具有不可或缺的重要作用。最新研究显示, 胚乳的整个基因组DNA甲基化水平降低, 而且去甲基化作用可能源于雌配子体的中央细胞。该文综述了种子发育的表观遗传学调控机制, 包括基因组印迹机制以及胚乳基因组DNA甲基化变化研究的最新进展。     

Female gametophyte development and double fertilization in Balsas teosinte, Zea mays subsp. parviglumis (Poaceae)

Over the course of maize evolution, domestication played a major role in the structural transition of the vegetative and reproductive characteristics that distinguish it from its closest wild relative, Zea mays subsp. parviglumis (Balsas teosinte). Little is known, however, about impacts of the domestication process on the cellular features of the female gametophyte and the subsequent reproductive events after fertilization, even though they are essential components of plant sexual reproduction. In this study, we investigated the developmental and cellular features of the Balsas teosinte female gametophyte and early developing seed in order to unravel the key structural and evolutionary transitions of the reproductive process associated with the domestication of the ancestor of maize. Our results show that the female gametophyte of Balsas teosinte is a variation of the Polygonum type with proliferative antipodal cells and is similar to that of maize. The fertilization process of Balsas teosinte also is basically similar to domesticated maize. In contrast to maize, many events associated with the development of the embryo and endosperm appear to be initiated earlier in Balsas teosinte. Our study suggests that the pattern of female gametophyte development with antipodal proliferation is common among species and subspecies of Zea and evolved before maize domestication. In addition, we propose that the relatively longer duration of the free nuclear endosperm phase in maize is correlated with the development of a larger fruit (kernel or caryopsis) and with a bigger endosperm compared with Balsas teosinte.     

Comparative embryology of basal angiosperms

Recent phylogenetic analyses of basal angiosperms have identified those lineages central to the study of the origin and early diversification of flowering plants. As we begin to understand the early evolution of endosperm developmental patterns in flowering plants, it is apparent that we know little about the other basic embryological features of basal angiosperms, such as the nature of the female gametophyte and even whether a process of double fertilization occurs.     

Microtubules in early development of the megagametophyte of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.     

The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis

Double fertilization of the female gametophyte produces the endosperm and the embryo enclosed in the maternal seed coat. Proper seed communication necessitates exchanges of signals between the zygotic and maternal components of the seed. However, the nature of these interactions remains largely unknown. We show that double fertilization of the Arabidopsis thaliana female gametophyte rapidly triggers sustained cell proliferation in the seed coat. Cell proliferation and differentiation of the seed coat occur in autonomous seeds produced in the absence of fertilization of the multicopy suppressor of ira1 (msi1) mutant. As msi1 autonomous seeds mostly contain autonomous endosperm, our results indicate that the developing endosperm is sufficient to enhance cell proliferation and differentiation in the seed coat. We analyze the effect of autonomous proliferation in the retinoblastoma-related1 (rbr1) female gametophyte on seed coat development. In contrast with msi1, supernumerary nuclei in rbr1 female gametophytes originate mainly from the endosperm precursor lineage but do not express an endosperm fate marker. In addition, defects of the rbr1 female gametophyte also reduce cell proliferation in the ovule integuments before fertilization and prevent further differentiation of the seed coat. Our data suggest that coordinated development of the seed components relies on interactions before fertilization between the female gametophyte and the surrounding maternal ovule integuments and after fertilization between the endosperm and the seed coat.     

Modularity of the angiosperm female gametophyte and its bearing on the early evolution of endosperm in flowering plants

The monosporic seven-celled/eight-nucleate Polygonum-type female gametophyte has long served as a focal point for discussion of the origin and subsequent evolution of the angiosperm female gametophyte. In Polygonum-type female gametophytes, two haploid female nuclei are incorporated into the central cell, and fusion of a sperm cell with the binucleate central cell produces a triploid endosperm with a complement of two maternal and one paternal genomes, characteristic of most angiosperms. We document the development of a four-celled/four-nucleate female gametophyte in Nuphar polysepala (Engelm.) and infer its presence in many other ancient lineages of angiosperms. The central cell of the female gametophyte in these taxa contains only one haploid nucleus; thus endosperm is diploid and has a ratio of one maternal to one paternal genome. Based on comparisons among flowering plants, we conclude that the angiosperm female gametophyte is constructed of modular developmental subunits. Each module is characterized by a common developmental pattern: (1) positioning of a single nucleus within a cytoplasmic domain (pole) of the female gametophyte; (2) two free-nuclear mitoses to yield four nuclei within that domain; and (3) partitioning of three uninucleate cells adjacent to the pole such that the fourth nucleus is confined to the central region of the female gametophyte (central cell). Within the basal angiosperm lineages Nymphaeales and Illiciales, female gametophytes are characterized by a single developmental module that produces a four-celled/four-nucleate structure with a haploid uninucleate central cell. A second pattern, typical of Amborella and the overwhelming majority of eumagnoliids, monocots, and eudicots, involves the early establishment of two developmental modules that produce a seven-celled/eight-nucleate female gametophyte with two haploid nuclei in the central cell. Comparative analysis of ontogenetic sequences suggests that the seven-celled female gametophyte (two modules) evolved by duplication and ectopic expression of an ancestral Nuphar-like developmental module within the chalazal domain of the female gametophyte. These analyses indicate that the first angiosperm female gametophytes were composed of a single developmental module, which upon double fertilization yielded a diploid endosperm. Early in angiosperm history this basic module was duplicated, and resulted in a seven-celled/eight-nucleate female gametophyte, which yielded a triploid endosperm with the characteristic 2:1 maternal to paternal genome ratio.     

Maternal Gametophyte Effects on Seed Development in Maize

Flowering plants, like placental mammals, have an extensive maternal contribution toward progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization that produces sister progeny, the endosperm and the embryo, of the seed. Because of this, there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays subsp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo, although some embryo-specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization, revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed.     

The four-celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): implications for understanding the origin and early evolution of monocots, eumagnoliids,and eudicots

The recent consensus that Amborellaceae, Nymphaeales, and Austrobaileyales form the three earliest-diverging lineages of angiosperms has led comparative biologists to reconsider the origin and early developmental evolution of the angiosperm seven-celled/eight-nucleate (Polygonum-type) female gametophyte. Illicium mexicanum (Illiciaceae; Austrobaileyales) develops a four-celled/four-nucleate female gametophyte. The ontogenetic sequence of the Illicium female gametophyte is consistent with that of all other Austrobaileyales and also with all Nymphaeales and is likely a plesiomorphy of angiosperms. A character analysis based on more than 250 embryological studies indicates that a transition from an ancestrally four-celled/four-nucleate Illicium-like female gametophyte to a seven-celled/eight-nucleate female gametophyte occurred in the common ancestor of the sister group to Austrobaileyales (a clade that includes monocots, eumagnoliids, and eudicots). Comparative analysis of reconstructed ancestral female gametophyte ontogenies identifies specific early stages of ontogeny that were modified during this transition. These modifications generated two important angiosperm novelties-a set of three persistent antipodal cells and a binucleate central cell, which upon fertilization yields a triploid endosperm. Early angiosperms are anatomically quite diverse in these two features, although triploid endosperm, composed of one paternal genome and two maternal genomes, is a conserved feature of the overwhelming majority of angiosperms.     

A developmental and evolutionary analysis of embryology in Platanus (platanaceae), abasal eudicot

The Platanaceae are an early derived eudicot lineage and therefore occupy a key position for understanding reproductive character diversification associated with the early evolutionary radiation of flowering plants. We conducted an embryological study of Platanus racemosa in order to provide critical data on defining angiosperm reproductive characters for this important group. Female gametophyte development is monosporic. Embryogenesis occurs in a series of stages including zygote elongation and division, development of a linear proembryo, formation of the embryo proper, histogenesis, organogenesis, and growth. Endosperm development is a complex process that includes four distinct phases: free nuclear proliferation, cellularization of the chalazal zone, centripetal cellularization of the micropylar zone, and cellular differentiation and growth. Only the outer endosperm layer persists at seed maturity. Our findings differ significantly from previously published reports for Platanus, in which endosperm development was described as ab initio cellular. A comparison of endosperm development in Platanus with several closely and distantly related free nuclear taxa reveals considerable developmental variability, consistent with a hypothesis of multiple origins of free nuclear endosperm in angiosperms. Our analysis indicates that much remains to be learned about embryology in basal angiosperms. Additional developmental and comparative studies will likely reveal critical insights into the early evolution of flowering plants.     

Double fertilization in flowering plants: 1898–2008

The present article gives a brief survey of results of studies in the area of plant embryology directly associated with the discovery made by S.G. Navashin in 1898 of double fertilization in vivo and in vitro. These studies utilized methods of electronic and fluorescence microscopy, cytophotometry, and cultures of isolated ovules, sperm, and the embryo sac central cell. Questions related to the origin of the female gametophyte of flowering plants, double fertilization, and the endosperm are considered. It is emphasized that progress in this field is associated chiefly with the study of molecular processes that regulate the development and functioning of the female gametophyte and the sporophyte on early stages of ontogenesis.     

Embryology of <Emphasis Type="Italic">Cardiopteris</Emphasis> (Cardiopteridaceae,Aquifoliales), with emphasis on unusual ovule and seed development

Cardiopteris (Cardiopteridaceae), a twining herb of two or three species distributed from Southeast Asia to Northern Australia, requires an embryological study for better understanding of its reproductive features. The present study of C. quinqueloba showed that the ovule and seed development involves a number of unusual structures, most of which are unknown elsewhere in angiosperms. The ovule pendant from the apical placenta is straight (not orthotropous), ategmic, and tenuinucellate, developing a monosporic seven-celled/eight-nucleate female gametophyte with an egg apparatus on the funicular side. Fertilization occurs by a pollen tube entering from the funicular side, resulting in a zygote on the funicular side. The endosperm is formed by the cell on the funicular side in the two endosperm cell stage. While retaining a (pro)embryo/endosperm as it is, the raphe (differentiating late in pre-fertilization stages) elongates toward the antiraphal side during post-fertilization stages, resulting in an anatropous seed. The two-cell-layered nucellar epidermis (belatedly forming by periclinal divisions), along with the raphe, envelops the embryo/endosperm entirely as the seed coat. The possibility was discussed that the arrested integument development triggers a series of the subsequent unusual structures of ovule and seed development. The fertilization mode in Cardiopteris underpins the hypothesis that the Polygonum?type female gametophyte comprises two four-celled archegonia.     

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.     

Development and function of central cell in angiosperm female gametophyte

The central cell characterizes the angiosperm female gametophyte (embryo sac or megagametophyte) in that it directly participates in “double fertilization” to initiate endosperm development, a feature distinguishing angiosperm from all other plant taxa. Polygonum‐type central cell is a binucleate cell that, upon fertilization with one of the two sperm cells, forms triploid endosperm to nourish embryo development. Although the formation and the structure of central cell have well been elucidated, the molecular mechanisms for its specification and development remain largely unknown. The central cell plays a critical role in pollen tube guidance during pollination and in endosperm initiation after fertilization. Recently, a group of mutants affecting specific steps of central cell development and function have been identified, providing some clues in understanding these questions. This review summarizes our current knowledge about central cell development and function, and presents overview about hypotheses for its evolution. genesis 48:466–478, 2010. © 2010 Wiley‐Liss, Inc.     

Kin selection and conflict in seed maturation

There are four genetically distinct components in the developing seeds of flowering plants: maternal sporophyte, gametophyte, endosperm, and embryo. Each component can potentially influence the quantity or quality of nutrients provided to the embryo of its seed, thereby reducing the amount available to embryos in other seeds of that plant. The theory of kin selection predicts that each component will be selected to favor its own embryo over the other embryos to the extent that it is more closely related to its own. Under this criterion, an embryo should be selected to try to acquire more nutrients than the endosperm should be selected to provide, the endosperm should try to supply more than the gametophyte should, and the gametophyte more than the parent sporophyte. Evidence for this conflict of interests is found in the higher frequency of endopolyploidy, nutrient-absorbing haustoria, and food storage tissues in the embryo and endosperm than in the gametophyte of maternal tissues.This theory also suggests how the gametophyte, which is the nurse tissue of gymnosperm seeds, was displaced from this role in the flowering plants by an endosperm initiated by a secondary fertilization. “Neoteny” in the pro-angiosperms created conditions in which (1) an endosperm initiated by double fertilization would be more closely related to the embryo than is the gametophyte and (2) the endosperm would be formed early enough to be of significant aid to the embryo.If this theory is correct it (1) requires a different approach to the study of seed morphology and physiology, (2) increases the plausibility of arguments that flowering plants are a polyphyletic group, (3) provides evidence that parents cannot always control the outcome of conflict with their offspring, and (4) forges a conceptual link in our understanding of the evolution of social interactions in plants and animals.     

Mutations in FIE, a WD polycomb group gene, allow endosperm development without fertilization

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif-containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.