《种子学》课程教学资源（文献资料）Fertilization and early seed formation

COMPTES RENDUS ScienceDirect ELSEVIER C.R.Biologies331(2008)715-725 BIOLOGIES http://france.elsevier.com/direct/CRASS3/ Review/Revue Fertilization and early seed formation Christian DumasPeter Rogowskyb. Available online 30 August 2008 Presented by Georges Pelletier Abstract The double fertilization of flowering plants is a complex process,encompassing multiple steps.From its discovery more than century ago,many useful descriptive approaches have been employed to better unveil specific steps/mechanisms.More recently. cially with the search of mut affected in the fertilization step llowed one to elucidate this critical phenomenon in living organisms.Genes invoved in pollen tube guidance or p lischarge in synergids have b identified,as well as genes e iting dimfe al express nism with an elaborate body plan.The development of the fertilized central cell intoa nourishing tissue (endosperm)starts with the formation of the coenocyte cell unique in th of the wsky.C.R.Biologies 331 (2008). 2008 Academie des sciences.Published by Elsevier Masson SAS.All rights reserved. Resume ment precoce de la graine La double fecc opeuvent presenter des limitations.La recherche de nouvelles strategies,en de mutant elle permet mieu prendre cetle et ue et unique de el-in rieure de Lyon 1.IFR1 BioSciences Lyon Gerland.Unite Rep n斤RR URL:http://www.ens-lyon fr/RDP/

C. R. Biologies 331 (2008) 715–725 http://france.elsevier.com/direct/CRASS3/ Review / Revue Fertilization and early seed formation Christian Dumas a,b,c , Peter Rogowsky a,b,c,∗ a Université de Lyon, École normale supérieure de Lyon, université Lyon 1, IFR128 BioSciences Lyon Gerland, Unité Reproduction et développement des plantes, 69364 Lyon, France b INRA, UMR879 Reproduction et développement des plantes, 69364 Lyon, France c CNRS, UMR5667 Reproduction et développement des plantes, 69364 Lyon, France Available online 30 August 2008 Presented by Georges Pelletier Abstract The double fertilization of flowering plants is a complex process, encompassing multiple steps. From its discovery more than a century ago, many useful descriptive approaches have been employed to better unveil specific steps/mechanisms. More recently, the development of an in vitro assay developed in our laboratory, has allowed a better understanding of this phenomenon. However, in vitro methods may show some limitations. The search for complementary strategies, especially with the search of mutants affected in the fertilization step allowed one to elucidate this critical and unique phenomenon in living organisms. Genes involved in pollen tube guidance or pollen discharge in synergids have been identified, as well as genes exhibiting differential expression in sperm, egg and central cells before and after fertilization. A calcium wave proved to correspond to the first cellular event seen after cytoplasmic fusion in the fertilized egg cell or zygote, which develops into a multi-cellular organism with an elaborate body plan. The development of the fertilized central cell into a nourishing tissue (endosperm) starts with the formation of the coenocyte, a multinuclear single cell unique in the plant kingdom, cellularization occurring later on. The balance of the paternal and maternal genomes, which is under the control of the FIS polycomb group complex, was found to be of the utmost importance for the successful development of the seed. To cite this article: C. Dumas, P. Rogowsky, C. R. Biologies 331 (2008). © 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved. Résumé Fécondation et développement précoce de la graine. La double fécondation des plantes à fleurs est un phénomène complexe comportant plusieurs étapes. Depuis sa découverte, il y a plus d’un siècle, plusieurs approches, essentiellement descriptives, ont été successivement développées. Plus récemment, une approche in vitro a permis de mieux comprendre ce phénomène. Néanmoins, de telles méthodes in vitro peuvent présenter des limitations. La recherche de nouvelles stratégies, en particulier l’étude de mutants affectés dans la fécondation, est utile car elle permet de mieux comprendre cette étape critique et unique des organismes vivants. Ces nouvelles approches ont permis la découverte de gènes impliqués dans l’attraction du tube pollinique ou la décharge du pollen dans les synergides ainsi que la caractérisation de gènes exprimés de manière différentielle entre le gamète mâle, l’oosphère et Abbreviations: CZE, chalazal endosperm; ESR, embyro surrounding region; MCE, micropylar endosperm; MGE, maternal genome excess; PEN, peripheral endosperm; PGE, paternal genome excess; NCD, nuclear cytoplasmic domain; PT, pollen tube; RMS, radial microtubule system; SI, self-incompatibility. * Corresponding author at: Université de Lyon, École normale supérieure de Lyon, université Lyon 1, IFR128 BioSciences Lyon Gerland, Unité Reproduction et développement des plantes, 69364 Lyon, France. E-mail address: peter.rogowsky@ens-lyon.fr (P. Rogowsky). URL: http://www.ens-lyon.fr/RDP/. 1631-0691/$ – see front matter © 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.crvi.2008.07.013

716 C.Dumas.P.Rogowsky/C.R.Biologies 331(2008)715-725 la cellule centrale avant ou apres fecondation.Une vague de calcium est le premier venement cellulaire documente apres fusion cpar le comppolycomb Fmrcnciale pour lesu d developpement de la grine.Pcrc s331(0 SAS.All rights reserved. Keywords:Gamete:Egg cell:Fertilization:Zygolelembryo;Endosperm:Angiosperms Mors-cles:Gamete:Oosphere:Fecondation:Zygote/embryon:Albumen:Angiospermes 1.Introduction tomogamy),wind (anemogamy)and,more rarely. Awye pblishedpecal of h -A progam es occu des science g or at the n Ci cell Mo ble fertilization process in plants.Thes neer authors he ee differ t pr cesses such as adhesion of the pollen independently observed for the first time the occurrence grain to pistil prior to water uptake,enzyme (e.g. of a double fertilization in two different plant species. cytochrome oxidase,cellulase.phosphorylase,ri- Turk's cap lily (Lilium martagon)and the perennial herb bonuclease,acid phosphatase,...)release and ac Fritillaria tenella.At that time no one was using model tivation,and the preparation of pollen tube (PT) s important discover formation.In thi s tube a second mitotic division b the on in plan ng to 0n0 que vehicle nd the establishment of a new ration.from the to their target cells within the so-caled"ule to the embryo included within the seed.The beg in (a terminology that is somehow misleading in re of embrvology research during the 20th century led to lation to animal or algae terminology).Ovules in many novel aspects in developmental biology.notably angiosperms are matemal organs containing the fe in plants.Sev ral books have been published on thi e monograph by Masheswari re ref related to em evolut ut ha eds of ve prevent self-fertilization.The mos 2.Fertilization in angiosperms:a multi-step cated and widespread of these mechanisms is self phenomenon incompatibility (SI).a process leading to the rejec tion of self-pollen by the pistil.SI is controlled by Fertilization in flowering plants (angiosperms)is a a single multiallelic locus,the S-locus (S standing very complex process as compared with animal or algae for self-incompat inty)and several plant systems systems.It consists of three successive phases (Fig.1): ave been carefully ana edfor this aspect.e.g. Papaver (I- Pollination that c to th tra of a hird p叫 of fe tophyte or pollen grain.from the male org the anther.to the receptive female organ,the stigma in angiosperms.Here.the first sperm nucleus fuses surface of the pistil.Note that since plants are ses- with the egg nucleus (at the origin of the zygotic sile organisms,sexual partners just meet by chance. embryo),while the second sperm nucleus fuses sometimes with the aid of animals like insects (en- with the two polar nuclei of the central cell of the

716 C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 la cellule centrale avant ou après fécondation. Une vague de calcium est le premier évènement cellulaire documenté après fusion cytoplasmique dans l’oosphère fécondé ou zygote, qui se développe en un organisme pluricellulaire avec un plan d’organisation très élaboré. Le développement de la cellule centrale en tissu nourricier, l’albumen, commence par la formation d’un cénocyte, une cellule multi-nucléée unique aux plantes, qui sera suivie par une cellularisation. La balance entre les génomes paternel et maternel, contrôlée par le complexe polycomb FIS, a une importance cruciale pour le succès du développement de la graine. Pour citer cet article : C. Dumas, P. Rogowsky, C. R. Biologies 331 (2008). © 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved. Keywords: Gamete; Egg cell; Fertilization; Zygote/embryo; Endosperm; Angiosperms Mots-clés : Gamète ; Oosphère ; Fécondation ; Zygote/embryon ; Albumen ; Angiospermes 1. Introduction A few years ago, we published a special issue of the Comptes rendus de l’Académie des sciences [1] to cel￾ebrate the centenary of the independent discovery by Sergius Nawashin [2] and Léon Guignard [3] of the dou￾ble fertilization process in plants. These pioneer authors independently observed for the first time the occurrence of a double fertilization in two different plant species, Turk’s cap lily (Lilium martagon) and the perennial herb Fritillaria tenella. At that time no one was using model plants or model systems. Yet, this important discovery still corresponds to one of the key hallmarks in plant biology. Indeed, the double fertilization is unique to flowering plants among living organisms and permits the establishment of a new generation, from the zygote to the embryo included within the seed. The beginning of embryology research during the 20th century led to many novel aspects in developmental biology, notably in plants. Several books have been published on this topic [4], of which the monograph by Masheswari re￾mains the historical reference in plant embryology [5]. In this paper, we summarize key features related to fer￾tilization and the earliest developmental aspects of em￾bryo tissues within the seeds of flowering plants. 2. Fertilization in angiosperms: a multi-step phenomenon Fertilization in flowering plants (angiosperms) is a very complex process as compared with animal or algae systems. It consists of three successive phases (Fig. 1): – Pollination that corresponds to the transfer of a male nucleus containing unit, called male game￾tophyte or pollen grain, from the male organ, the anther, to the receptive female organ, the stigma surface of the pistil. Note that since plants are ses￾sile organisms, sexual partners just meet by chance, sometimes with the aid of animals like insects (en￾tomogamy), wind (anemogamy) and, more rarely, water (hydrogamy). – A progamic phase that includes all processes occur￾ring from the landing of pollen grains on the recep￾tive stigma to the time that the sperm cell reaches the egg cell. More precisely, this phase includes different processes such as adhesion of the pollen grain to pistil prior to water uptake, enzyme (e.g., cytochrome oxidase, cellulase, phosphorylase, ri￾bonuclease, acid phosphatase, . . . ) release and ac￾tivation, and the preparation of pollen tube (PT) formation. In this tube, a second mitotic division often occurs leading to the formation of two sperm cells. Here, the PT acts as a sort of vehicle (sperm cells are not motile) carrying the two sperm cells to their target cells within the so-called “ovule” (a terminology that is somehow misleading in re￾lation to animal or algae terminology). Ovules in angiosperms are maternal organs containing the fe￾male gametophyte, the embryo sac (a complex hap￾loid and pluricellular structure containing two fe￾male gametes), the egg cell and the central cell (Fig. 1). During the course of evolution, about half of the angiosperm species have acquired specific recognition mechanisms, which strongly limit or even prevent self-fertilization. The most sophisti￾cated and widespread of these mechanisms is self￾incompatibility (SI), a process leading to the rejec￾tion of self-pollen by the pistil. SI is controlled by a single multiallelic locus, the S-locus (S standing for self-incompatibility) and several plant systems have been carefully analyzed for this aspect, e.g., Brassica or Papaver [7–9]. – The third phase, referred to as syngamy, is the last and decisive phase of fertilization, which corre￾sponds to the unique double nuclear fusion event in angiosperms. Here, the first sperm nucleus fuses with the egg nucleus (at the origin of the zygotic embryo), while the second sperm nucleus fuses with the two polar nuclei of the central cell of the

C.Dumas,P Rogowsky/C.R.Biologles 331(2008)715-725 71 Pollen hydration germination Incompatible situation Pollination Self pollen Compatible situation Non self pollen Progamic phase ovule embryo sac ovary Fig1.The steps of fertilization in flowering plants. embryo to form the triploid endosperm(Fig.2). (Zea mays)was used to isolate both male and fe These three steps.and especially the syngamy step. male gametes (sperm,egg and central cells)[12] are now well described and largely understood both to establish an in vitro system mimicking in vivo at the cytological and molecular levels since the conditions [1 3].Caryogamy was clearly examined mb between an in viro assa on n cover and I m cEctonmi using the (2.3).Lat an incr eve in the ad as oceurs in animal o nete fusion [15].This rise oc its use to elucidate the s notably with the curred after the establishment of gamete cytoplasm continuity.With the aid of an extracellular vibrat- These crucial observations made by the group of ing probe,this allowed one to demonstrate that a w Jensen first showed that the two male gametes calcium influx is triggered and propagates in the zy- are real cells devoid of cell walls [10].Second gote as a wave front [16]. they documented that the plasma membrane of the gamete cells is in close contact with the inner 3.Molecular and cellular biology of fertilizatior plasma membrane of the vegetative cell that sur- rounds them in the pollen the The question arises as to whether Caz+accumula tion is necessary and/or su ent to trigger egg act cture,forming the and the initiation of deve The vitro approaches could

C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 717 Fig. 1. The steps of fertilization in flowering plants. embryo to form the triploid endosperm (Fig. 2). These three steps, and especially the syngamy step, are now well described and largely understood both at the cytological and molecular levels since the original description by Nawashin and Guignard [6,7]. Lilium martagon and Fritillaria tenella were originally used to discover and first describe the double fertilization process using the classical mi￾croscope [2,3]. Later, a new source of excitement came with the advent of electron microscopy and its use to elucidate the syngamy, notably with the aid of cotton (Gossypium hirsutum) as a model. These crucial observations made by the group of W. Jensen first showed that the two male gametes are real cells devoid of cell walls [10]. Second, they documented that the plasma membrane of the gamete cells is in close contact with the inner plasma membrane of the vegetative cell that sur￾rounds them in the pollen grain. In addition, the nucleus of the vegetative cell seems to be physically associated to this structure, forming the so-called ‘male germ unit’ [11]. In more recent studies, maize (Zea mays) was used to isolate both male and fe￾male gametes (sperm, egg and central cells) [12] to establish an in vitro system mimicking in vivo conditions [13]. Caryogamy was clearly examined by using a combination between an in vitro assay and a three-dimensional image reconstruction from electron microscopy data [14]. The first detectable cellular event taking place after gamete fusion was an increase in the concentration of cytosolic Ca2+, as occurs in animal gamete fusion [15]. This rise oc￾curred after the establishment of gamete cytoplasm continuity. With the aid of an extracellular vibrat￾ing probe, this allowed one to demonstrate that a calcium influx is triggered and propagates in the zy￾gote as a wave front [16]. 3. Molecular and cellular biology of fertilization The question arises as to whether Ca2+ accumula￾tion is necessary and/or sufficient to trigger egg acti￾vation and the initiation of development. The sole use of in vitro approaches could not help answering this

718 C.Dumas.P.Rogowsky C.R.Biologies 331 (2008)715-725 Pollen tuh Zvoote (ntn Pollen Gametic cell Caryogamy Embryo tube fusion development scharge and shown to encode a DNAJ chap onin localized to the mite ndria.Thus.most of eDNA libraries prepared from isolated gametes i7 dea depends on the invo and zygotes [18].(b)new developments including life ntal ablation studies inth imaging using Arabidopsis (Arabidopsis thaliana)as a ia f ieri)sug est tha model system [19,20].and (c)the search for mutants only intact synergids can attract the PT to the underscoring the impor tance of hoth syn ergids in PT sperm-co rmation to be receint Here the rsistent synergid is important for ru attraction and the other typically de enerated receptive s In fact h ery rabid synergid is important for entry of the Pr in the em onment is not affected and bryo sac [25].A critical role for actin coronas has also synergid differentiation appears to be normal.However. been observed in degenerated synergids.both near the after pollen tube penetration in the embryo sac a failure egg nucleus and ending near the central cell [26].Also. a myb protein has been identified as a transcriptional in sperm discharge is evidenced [22].After the recent characterization of FER/SIR (FERONIA/SIRENE)as regulator of genes expressed in synergid cells and re quired for the formation of the filiform apparatus and ceptor kir PT guidance 271.Furthermore,the highly expressed pu or PT reception ha emerged [23.n Generative Cell Specificl(GCS1)protein located in the sperm cell membrane proved indispensable for the fu- sion of the two sperm cells with the egg cell and the naling cascade that enables the femaleg byte to central cell [28].Only fertilized eggs proved to be asso- prepare itself for fertilization.It is noted that in the ciated with actin coronas.in marked contrast to unfertil- gametophytic factor2 mutant the synergids do not de- ized supernumerary egg cells lacking an actin corona as generate while they normally do so during the fertiliza- occur in the maize mutant indeterminate gametophytel tion process.The corresponding gene has been cloned (ig)[291

718 C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 Fig. 2. Events of double fertilization. The interaction between the male (pollen tube) and female (embryo sac) gametophyte is followed by gametic cell fusion, caryogamy and embryonic development. question. Therefore, several complementary approaches have been developed including (a) molecular analyses of cDNA libraries prepared from isolated gametes [17] and zygotes [18], (b) new developments including life imaging using Arabidopsis (Arabidopsis thaliana) as a model system [19,20], and (c) the search for mutants affected in fertilization. This allowed a novel class of MYB factors controlling sperm-cell formation to be identified [21]. Furthermore, the female gamete was found to be able to regulate the delivery of male ga￾metes. In fact, in the feronia and sirene Arabidopsis mutants, embryo sac development is not affected and synergid differentiation appears to be normal. However, after pollen tube penetration in the embryo sac a failure in sperm discharge is evidenced [22]. After the recent characterization of FER/SIR (FERONIA/SIRENE) as a synergid-expressed, plasma-membrane-localized re￾ceptor kinase, a putative model for PT reception has emerged [23]. In this model, when the pollen tube reaches the synergids, a ligand issued from the PT binds to the FER/SIR extracellular domain, triggering a sig￾naling cascade that enables the female gametophyte to prepare itself for fertilization. It is noted that in the gametophytic factor2 mutant the synergids do not de￾generate while they normally do so during the fertiliza￾tion process. The corresponding gene has been cloned and shown to encode a DNAJ chaperonin localized to the mitochondria. Thus, most presumably synergid cell death depends on the involvement of some mitochon￾drial function [24]. Experimental ablation studies in the plant Wishbone flowers (Torenia fournieri) suggest that only intact synergids can attract the PT to the ovule, underscoring the importance of both synergids in PT receipt. Here, the persistent synergid is important for attraction and the other typically degenerated receptive synergid is important for entry of the PT in the em￾bryo sac [25]. A critical role for actin coronas has also been observed in degenerated synergids, both near the egg nucleus and ending near the central cell [26]. Also, a MYB protein has been identified as a transcriptional regulator of genes expressed in synergid cells and re￾quired for the formation of the filiform apparatus and PT guidance [27]. Furthermore, the highly expressed Generative Cell Specific1 (GCS1) protein located in the sperm cell membrane proved indispensable for the fu￾sion of the two sperm cells with the egg cell and the central cell [28]. Only fertilized eggs proved to be asso￾ciated with actin coronas, in marked contrast to unfertil￾ized supernumerary egg cells lacking an actin corona as occur in the maize mutant indeterminate gametophyte1 (ig1) [29]

C.Dumas,P Rogowsky/C.R.Biologies 331(2008)715-725 719 Nuclear migration within the central cell generally tion of an apico-basal polarity,the differentiation of an precedes fertilization.Furthermore,egg cell fertiliza- epidermis and the formation of the shoot and root mers- on being whe e (ccg). 31.The o in vith an N-t of the ner the ences between a cell rich in eytoplasm.which giv terchangeable with that of tellb (a basal transerintion to the embryo proper and a strongly vacuolized cell at factor)in yeast,suggesting that CCG might act as a tran- the origin of the suspensor [41].Marker genes specif scription regulator for PT guidance. ically expressed in either cell,such as WoX2 in the ops ryo 42 tion has ad. the piar h and in B ch rimental data sur ort the rence of preferential fertilization as ue first div general phe- ing that polarity is already established in the zygot nomenon 1341. egg cell [35].In this context it is interesting to note that a rather dramatic shift in cell polarity takes place in 4.Early embryo and endosperm development the maize egg cell upon fertilization.While the nucleus The of the egg ce to f they are I centra to p waCae ung th rate body plans ely the embr o and the endo is L.)[43.441.This observ os in are generally referred to as early deve ment This de. favor of an influence of the fertilization p itself velopment provides the structures necessary for the ac- on the polarity of the zygote.The remaining events of cumulation of reserve storage molecules,which in the early embryo development are discussed elsewhere in case of the embryo are used for further development af- this issue 45. ter germination. The development of the endosperm starts with the t embryos is fundamentally B opment.Both o miniature (Ar of the (ba containing at least primordia of all its future though it is believed that this endosperm ar ind It is rather the beginning of a continuous developmen- pendently during evolution in these two lineages [46 tal process interrupted temporarily by drying and dor- In nuclear endosperm,the initial endosperm nucleus di mancy [35].Secondly,the elaboration of the body plan vides repeatedly without cell wall formation,resulting is not based on cell lineage but on the position of in- in a coenocytic endosperm,in which the nuclei are dis dividual cells within ryo 361.gradients of hor ing a central vacuol 1g.3 stage 1 oplast orthe phase band [38] m from cell fusion rather thar Finally.the cells of the plant embryo are not mobile from nuclear division.The nuclear divisions are highly and show neither the long-distance homing nor the in- synchronized resulting in arabidonsis in over 200 nu terstitial migration seen in animal and in particular in clei after eight rounds of division.While the first three vertebrate embryos [39]. rounds of division are fully synchronized,a slight delay of some divisions during the following rounds allows evelopment is mar ition of three mi major events comresponding respectively to the ing synchron micropylar en

C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 719 Nuclear migration within the central cell generally precedes fertilization. Furthermore, egg cell fertiliza￾tion precedes central cell fertilization both in Arabidop￾sis and maize, two well studied plant models [19,30]. Central cell guidance (ccg), a new mutant defective in micropylar PT guidance has been identified [31]. The CCG gene encodes a nuclear protein with an N-terminal conserved zinc β-ribbon domain that is functionally in￾terchangeable with that of TFIIB (a basal transcription factor) in yeast, suggesting that CCG might act as a tran￾scription regulator for PT guidance. To date there are no reports pointing toward a sperm cell dimorphism in Arabidopsis. In fact, this exceptional phenomenon suggesting a preferential fertilization has only been reported in Ceylon Lead wort (Plumbago zey￾lanica) and in B chromosome line of maize [32,33]. However, new experimental data support the occur￾rence of preferential fertilization as a general phe￾nomenon [34]. 4. Early embryo and endosperm development The developmental events leading from the two sin￾gle cells, the zygote and the fertilized central cell, to two multi-cellular, highly differentiated organs with elabo￾rate body plans, namely the embryo and the endosperm, are generally referred to as early development. This de￾velopment provides the structures necessary for the ac￾cumulation of reserve storage molecules, which in the case of the embryo are used for further development af￾ter germination. The development of plant embryos is fundamentally different from that in animal systems. Firstly, plant em￾bryogenesis is not a distinct process leading to the for￾mation of a miniature version of the adult organism containing at least primordia of all its future organs. It is rather the beginning of a continuous developmen￾tal process interrupted temporarily by drying and dor￾mancy [35]. Secondly, the elaboration of the body plan is not based on cell lineage but on the position of in￾dividual cells within the embryo [36], gradients of hor￾mones or other signaling molecules determining the fate of individual cells [37]. Thirdly, plants have a unique mode of cytokinesis involving plant-specific structures such as the phragmoplast or the preprophase band [38]. Finally, the cells of the plant embryo are not mobile and show neither the long-distance homing nor the in￾terstitial migration seen in animal and in particular in vertebrate embryos [39]. In both monocots (e.g., maize) and dicots (e.g., Ara￾bidopsis) early embryo development is marked by three major events corresponding respectively to the acquisi￾tion of an apico-basal polarity, the differentiation of an epidermis and the formation of the shoot and root meris￾tem [40]. The origin of apico-basal polarity is a matter of debate, the question being whether this polarity is es￾tablished de novo in the embryo or inherited from the egg cell. Polarity is clearly established after the first cell division of the embryo due to clear cytological differ￾ences between a cell rich in cytoplasm, which gives rise to the embryo proper and a strongly vacuolized cell at the origin of the suspensor [41]. Marker genes specif￾ically expressed in either cell, such as WOX2 in the upper cell and WOX8 in the lower cell, support the po￾larization of the two-celled embryo [42]. While some authors claim that the plane of the first division, which is perpendicular to the embryo axis, is at the origin of embryo polarity [40], others argue that exceptions with longitudinal or oblique first divisions exist imply￾ing that polarity is already established in the zygote or egg cell [35]. In this context it is interesting to note that a rather dramatic shift in cell polarity takes place in the maize egg cell upon fertilization. While the nucleus and most of the cytoplasm are located at the micropylar half of the egg cell prior to fertilization, they are found in the antipodal half afterwards, matching the polarity of Arabidopsis or the plant shepherd’s purse (Capsella bursa-pastoris L.) [43,44]. This observation argues in favor of an influence of the fertilization process itself on the polarity of the zygote. The remaining events of early embryo development are discussed elsewhere in this issue [45]. The development of the endosperm starts with the fertilized central cell, which can undergo a cellular, nu￾clear or mixed (helobial), type of development. Both the Brassicaceae (Arabidopsis) and the Gramineae (barley, maize) have an endosperm of the nuclear type, even though it is believed that this endosperm arose inde￾pendently during evolution in these two lineages [46]. In nuclear endosperm, the initial endosperm nucleus di￾vides repeatedly without cell wall formation, resulting in a coenocytic endosperm, in which the nuclei are dis￾tributed in the periphery of a giant single cell, surround￾ing a central vacuole (Fig. 3). While this stage is also referred to as syncytial endosperm, we wish to avoid this term, because it implies for certain readers that the mult￾inucleate cytoplasm arose from cell fusion rather than from nuclear division. The nuclear divisions are highly synchronized resulting in Arabidopsis in over 200 nu￾clei after eight rounds of division. While the first three rounds of division are fully synchronized, a slight delay of some divisions during the following rounds allows the definition of three mitotic domains. While nuclei are still dividing synchronously within the micropylar en-