《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-

720 C.Dumas.P.Rogowsky C.R.Biologies 331 (2008)715-725 RMS R 2 nuclei8nuclei 128 nuclei alveolar cellularized phr RMS、 □embryo:embryo prop embryo:suspensor endosperm:ESR/MCE endosperm:starchy endospem/PEN 日Ee2x Fig.3.Early embryo and endosperm development in maize and Arabidopsis.Schematic drawings of embryo and en m in maize(op）an Sy (y)re no BET dosperm(MCE)surrounding the embryo and within the mation of"free growing"phragmoplasts at the zones peripheral endosperm(PEN)in the central chamber,no where growing NCDs contact each other.The third one more divisions are observed in the chalazal endosperm s alveolar cell w vall formation leading to a tube around (CZE)[47].Specific marker lines with domain-specific s with its open end toward the central v expressio lization of the endosperm the pericinal cell genes nav to be on pr of the ing through the PEN and CZE at different rates and and the MADS box trans Ctors AGL62 [52] with significant variations between the domains i5ol In maize,barley (Hordeum e)and rice (Orvz The first step of cellularization is the formation of a sativa)the early steps of endosperm development and in radial microtubule system(RMS)emanating from the particular the formation of the coenocyte and the mech surface of endosperm nuclei and defining nuclear cy anisms of early cellularization are very similar to Ara- toplasmic domains (NCD).The second one is the for- bidopsis [53,541.despite major morphological differ

720 C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 Fig. 3. Early embryo and endosperm development in maize and Arabidopsis. Schematic drawings of embryo and endosperm in maize (top) and Arabidopsis (bottom) at 5 stages: 2, 8 and 128 nuclei in the endosperm, alveolar and cellularized. Note that not all nuclei are in the plane presented by the drawing. The central vacuole (cv) of the single celled endosperm coenocyte is indicated in light blue at two stages. The radial microtubule system (RMS) is indicated around free nuclei. One of the alveoli (alv) is surrounded by a dotted white line. Synergids (sy) are not colored. BETL, basal endosperm transfer layer; CZE, chalazal zone endosperm; ESR, embryo surrounding region; MCE: micropylar cellularized endosperm; PEN, peripheral or central endosperm; phr, phragmoplast. dosperm (MCE) surrounding the embryo and within the peripheral endosperm (PEN) in the central chamber, no more divisions are observed in the chalazal endosperm (CZE) [47]. Specific marker lines with domain-specific expression confirm the regionalization of the endosperm prior to cellularization [48,49]. The cellularization process starts at the final round of coenocytic mitosis as a wave in the MCE, progress￾ing through the PEN and CZE at different rates and with significant variations between the domains [50]. The first step of cellularization is the formation of a radial microtubule system (RMS) emanating from the surface of endosperm nuclei and defining nuclear cy￾toplasmic domains (NCD). The second one is the for￾mation of “free growing” phragmoplasts at the zones where growing NCDs contact each other. The third one is alveolar cell wall formation leading to a tube around each nucleus with its open end toward the central vac￾uole. The last one is the formation of a periclinal cell wall at the next nuclear mitosis [50]. Several genes have been shown to be involved in the cellularization process, including members of the FIS polycomb group [51] and the MADS box transcription factors AGL62 [52]. In maize, barley (Hordeum vulgare) and rice (Oryza sativa) the early steps of endosperm development and in particular the formation of the coenocyte and the mech￾anisms of early cellularization are very similar to Ara￾bidopsis [53,54], despite major morphological differ-

C.Dumnas,P.Rog vsky/C.R.Biologies 331(2008)715-725 ences between the embryo surrounding region [55,56] crosses involving the indeterminate gametophyte (ig) the basal endosperm transfer layer [57,58],the starchy mutation in maize,which allowed to rule out a role of PEN.CZE i A and the Tunctonal equivalents MCE total ploidy levels or of the genom ratios between em (1g. the coat [66 ellulariza ops bryo.en osperm and Depending on terploid bit eith seny ca 2m:2p).Phen endosperm is consumed rapidly by the developing em- analyses of Arabidopsis seeds obtained by recipro bryo,while the cereal endosperm starts to accumulate crosses between diploid and tetraploid as well as be starch and protein re serves,which will nourish the em tween diploid and hexaploid line s showed that MGE and bryo PGE produce complementary phenotype Thus,MGE The e ced.m sperm c evel nt by pre re cellu ut th tw thth ed coat nd d embry o to the endos leadi to la s [671.Sim lar results weree ntly obtained in maize.It app mutant.which shows a strong paternal effect.In mu that MGE seeds are very small and generally abort, tant pollen,only one sperm cell,instead of two,is pre their endosperms cellularize earlier,they enter earlie duced The pollen is viable but can fertilize only the into endoreduplication and they accumulate significant egg cel and no e un In contrast, PGE seeds are slightly the 621.Evid fores pre n extende nd the s little starch 168.691.The comes from the analysis of the s Haiku?(ku? nisms mlation of the ont and Miniseed3 (Mini3)encoding a leucine-rich-repeat kinase and a transcription factor of the WRKY family. ing the balance between cell proliferation and cell dif respectively [63].Both mutants exhibit decreased en ferentiation are not known.However,there is agree dosperm size,along with decrease in cell elongation ment that parenta may play an importan in the seed integuments 64 role [51].Firs r this come om a s ma ern al genome in h maternal allelic sion dur arly ende n de Seed development is strongly influenced by the bal velopment perfectly correlates with methylation of the ance of the paternal and maternal genomes.In wild type 5'end of the paternal alleles.There was no Fie2 methy- seeds the diploid embryo generally contains one ma lation in sperm cell suggesting that the methylation ternal and one paternal genome (1m:Ip).In contrast was actively established during or shortly after fertil- an inbuilt al e analysi ations also seem to e in pater m diploid es b H3K9m I that the 1 het defects o the deve matin and inte the endosperm seems more affected than the embrvo by rochromatin concerned predominantly the maternal but developmental aberrations [5].While beneficial effects not the paternal genome [71.In addition,clustering of interploidy crosses on seed development have been of the HTR12 centromeric histone of paternal origin a documented in certain species. they remain an excep one extremity of endosperm nuclei even after two di tion [65].The first conclusive evidence for the impor visions was interpreted as a segregation of the paterna whicn may pre seed rom interploidy cpomcmt figure parent imprinting 72

C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 721 ences between the embryo surrounding region [55,56], the basal endosperm transfer layer [57,58], the starchy endosperm [59] and their functional equivalents MCE, PEN, CZE in Arabidopsis (Fig. 3). After cellularization the Arabidopsis endosperm en￾ters directly into endoreduplication, while numerous additional cell divisions occur in cereals prior to en￾doreduplication [60]. Following endoreduplication the endosperm is consumed rapidly by the developing em￾bryo, while the cereal endosperm starts to accumulate starch and protein reserves, which will nourish the em￾bryo after germination, as discussed in this issue [61]. The endosperm and embryo develop in parallel to form the seed, but little is known about the coordination between these two organisms as well as the coordina￾tion with the surrounding seed coat. Signaling from the embryo to the endosperm was suggested by the charac￾terization of the Arabidopsis cdc2A (also called cdka;1) mutant, which shows a strong paternal effect. In mu￾tant pollen, only one sperm cell, instead of two, is pro￾duced. The pollen is viable but can fertilize only the egg cell and not the central cell. However, the unfertil￾ized endosperm develops, suggesting that a previously unforeseen signal from the fertilized egg initiates pro￾liferation of the central cell [62]. Evidence for com￾munication between the endosperm and the seed coat comes from the analysis of the genes Haiku2 (Iku2) and Miniseed3 (Mini3) encoding a leucine-rich-repeat kinase and a transcription factor of the WRKY family, respectively [63]. Both mutants exhibit decreased en￾dosperm size, along with a decrease in cell elongation in the seed integuments [64]. 5. Balance of paternal and maternal genome contribution to embryo and endosperm Seed development is strongly influenced by the bal￾ance of the paternal and maternal genomes. In wild type seeds the diploid embryo generally contains one ma￾ternal and one paternal genome (1m:1p). In contrast, the triploid endosperm has an inbuilt imbalance of two maternal to one paternal genome (2m:1p). Deviation from these ratios, for example in interploidy crosses be￾tween diploid and tetraploid lines, frequently leads to defects or abortion of the developing seeds. Generally the endosperm seems more affected than the embryo by developmental aberrations [5]. While beneficial effects of interploidy crosses on seed development have been documented in certain species, they remain an excep￾tion [65]. The first conclusive evidence for the impor￾tance of the balance of maternal to paternal genomes for normal seed development came from interploidy crosses involving the indeterminate gametophyte (ig) mutation in maize, which allowed to rule out a role of total ploidy levels or of the genome ratios between em￾bryo, endosperm and the seed coat [66]. Depending on the direction of the interploidy cross, the progeny can exhibit either maternal genome excess (MGE, embryo: 2m:1p, endosperm: 4m:1p) or paternal genome excess (PGE, embryo: 1m:2p, endosperm: 2m:2p). Phenotypic analyses of Arabidopsis seeds obtained by reciprocal crosses between diploid and tetraploid as well as be￾tween diploid and hexaploid lines showed that MGE and PGE produce complementary phenotypes. Thus, MGE inhibits endosperm development by premature cellular￾ization, causing in turn a developmental delay or arrest of the embryo. In contrast, PGE promotes growth of the endosperm by accelerated mitoses and delayed cel￾lularization, leading to larger embryo sizes [67]. Simi￾lar results were recently obtained in maize. It appears that MGE seeds are very small and generally abort, their endosperms cellularize earlier, they enter earlier into endoreduplication and they accumulate significant amounts of starch. In contrast, PGE seeds are slightly bigger than MGE seeds though they are still smaller than wild type seeds, undergo an extended period of cell proliferation, show little endoreduplication and ac￾cumulate little starch [68,69]. The molecular mecha￾nisms causing the temporal deregulation of the onto￾genic program of endosperm development and disturb￾ing the balance between cell proliferation and cell dif￾ferentiation are not known. However, there is agree￾ment that parental imprinting may play an important role [51]. First evidence for this comes from methyla￾tion studies of the Fie1 (Fertilization independent en￾dosperm1) and Fie2 genes in isolated gametes and fer￾tilization products of maize showing that the exclusively maternal allelic expression during early endosperm de￾velopment perfectly correlates with methylation of the 5 end of the paternal alleles. There was no Fie2 methy￾lation in sperm cells, suggesting that the methylation was actively established during or shortly after fertil￾ization [70]. Histone modifications also seem to be in￾volved since the analysis of endosperm with altered parental genome dosage indicated that the redistribution of the H3K9me1 heterochromatin marker from chro￾mocenters toward euchromatin and interspersed hete￾rochromatin concerned predominantly the maternal but not the paternal genome [71]. In addition, clustering of the HTR12 centromeric histone of paternal origin at one extremity of endosperm nuclei even after two di￾visions was interpreted as a segregation of the paternal chromatin from the maternal chromatin, which may pre- figure parental genomic imprinting [72].

722 C.Dumas.P.Rogowsky C.R.Biologies 331 (2008)715-725 6.Development of unfertilized male and female ulation of the genes encoding members of the prc2 gametes is complex.Thu s.MEA does not only auto -regulate its own transcription 86 but is also subject to pater Unfertilized gametes can develop to certain degrees nal imprinting [87].just like certain other members and even give rise to haploid,generally sterile plants of the PRC2 complex.On the other hand,the target Artificial doubling of chromo omes at the seedling Pheres/is one of the rare genes known to be stage pre ents sterility and together with self pollinatior of perfectly homozy ent The de of the s.The fr all is generally very low.e.g.0.1%in maize 731.This fre eles [8ol More r ntly viahle seeds c quency can be dramatically increased by the use of par normal diploid embryo and an unfertilized endosperm ticular male parents (inducing lines)to reach for exam have been obtained by fertilization of prc2 mutants by ple 8.1%in maize [74].While unfertilized sperm cells hemizygous CDKA:/-YFP plants generating only on do not develop into embryos,immature m rospores the prod n hap :d by vanc ing the contribution of the hich ca n and has beer ated int rtile bng,This。 the perm rep ed and and more recently micros erary ciency is strongly species and genotype dependent and can reach over 30%in rapeseed [75].There are no re. References ports on substantial development of unfertilized central cells other than in mutants mu allowing the devel ☒s.G.Naw in.Resultate einer Revi Pol me (PcG i tene.Bul Acad which in wild phyte de d.Sur nent after fer tilization 176.771.In the seed the mplex is com (1899864-871 posed of the SET(Suppressor of variegat on/Enhancer 4]B.M zeste/Trithorax)domain protein MEDEA (MEA) 19g IZATION IN Embryology of Angiosperms.McGraw-Hill. (I [6]J.E Faure,C.Dumas,Fertilization in ring plants:New ap ctio hanisn in:B.R Jordan (Ed.).Th of members of th ment of the tral cell in the absence of fertilization CABI Publishing.2007.pp.269-297 8( In addition,msil mutants also show a few divisions of S.D.Russell.The g loid embryo The Polycomb Repressive Complex. (PRC2)is thougl at K2,gene [10]W.A.Jer en,D.B.Fisher.C tton embrvose is:the sperm on b methy 3K2 for thr a MADS Maize as box transcription factor [831.Fusca3 (Fus3)coding fo Cl( a B3 transcription domain factor [84]and Formin Ho 1337-1348. mology Protein5(FH5)encoding an actin nucleator [13】J.EF involved in endosperm cellularization [851.The reg. 1600

722 C. Dumas, P. Rogowsky / C. R. Biologies 331 (2008) 715–725 6. Development of unfertilized male and female gametes Unfertilized gametes can develop to certain degrees and even give rise to haploid, generally sterile plants. Artificial doubling of chromosomes at the seedling stage prevents sterility and together with self pollination allows the production of perfectly homozygous plants, which is of a great interest for plant breeders. The de￾velopment of the non-fertilized egg cell or gynogenesis naturally occurs in most Angiosperms. The frequency is generally very low, e.g. 0.1% in maize [73]. This fre￾quency can be dramatically increased by the use of par￾ticular male parents (inducing lines) to reach for exam￾ple 8.1% in maize [74]. While unfertilized sperm cells do not develop into embryos, immature microspores, the product of male meiosis, can be induced by various stresses to form haploid embryos in vitro, which can be regenerated into fertile plants after chromosome dou￾bling. This process was originally termed androgenesis and more recently microspore embryogenesis. Its effi- ciency is strongly species and genotype dependent and can reach over 30% in rapeseed [75]. There are no re￾ports on substantial development of unfertilized central cells other than in mutants. The best characterized mutations allowing the devel￾opment of unfertilized central cells concern members of the chromatin remodeling Polycomb group (PcG), which in wild type plants plays a major role in the arrest of female gametophyte development after fer￾tilization [76,77]. In the seed, the complex is com￾posed of the SET (Suppressor of variegation/Enhancer of zeste/Trithorax) domain protein MEDEA (MEA) [78], the VEFS domain protein FERTILIZATION IN￾DEPENDENT SEED2 (FIS2) [79], the WD40 re￾peat proteins FERTILIZATION INDEPENDENT EN￾DOSPERM (FIE) [80] and MULTICOPY SUPPRES￾SOR OF IRA1 (MSI1) [81]. Loss-of-function mutants of members of the complex show autonomous develop￾ment of the central cell in the absence of fertilization. In addition, msi1 mutants also show a few divisions of the egg cell leading to early arrested non-viable hap￾loid embryos[82]. The Polycomb Repressive Complex2 (PRC2) is thought to repress gene expression by methy￾lation of histone H3 at K27 (H3K27). Correlation of gene repression with the presence of the H3K27 marks has been demonstrated for three direct targets of the seed PRC2, namely Pheres1 (Phe1) coding for a MADS box transcription factor [83], Fusca3 (Fus3) coding for a B3 transcription domain factor [84] and Formin Ho￾mology Protein5 (FH5) encoding an actin nucleator involved in endosperm cellularization [85]. The reg￾ulation of the genes encoding members of the PRC2 is complex. Thus, MEA does not only auto-regulate its own transcription [86] but is also subject to pater￾nal imprinting [87], just like certain other members of the PRC2 complex. On the other hand, the target gene Pheres1 is one of the rare genes known to be subject to maternal imprinting by the PRC2 [88]. Ara￾bidopsis Glauce (Glc), whose molecular identity is not yet known, counterbalances the action of the PRC2 by promotion of fertilization-independent endosperm development and expression of paternally inherited al￾leles [89]. More recently, viable seeds consisting of a normal diploid embryo and an unfertilized endosperm have been obtained by fertilization of PRC2 mutants by hemizygous CDKA;1-YFP plants generating only one sperm cell. This result suggests a more general role of the PRC2 complex in balancing the contribution of the paternal genome in the triploid endosperm and has been used as an additional argument that the endosperm rep￾resents in evolution an extension of female gametophyte development rather than a supernumerary embryo [90]. References [1] C. Dumas, Reproduction et développement des plantes à fleurs, C. R. Acad. Sci. Paris, Ser. III 324 (2001) 517–521. [2] S.G. Nawashin, Resultate einer Revision der Befruchtungsvor￾gaenge bei Lilum Martagon und Fritillaria tenella, Bul. Acad. Imp. Sci. St. Petersburg 9 (1898) 377–382. [3] M.L. Guignard, Sur les anthérozoïdes et la double copulation sexuelle chez les végétaux angiospermes, C. R. Acad. Sci. 128 (1899) 864–871. [4] B.M. Johri, K.B. 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C.Dumas.,P.Rog wsky/C.R.Biologies 331(2008)715-725 [14]J.E.Faure,H.L Mogensen,C.Dumas,H.Lorz,E.Kranz. [34]J.E.Faure,M.L.Rusche,A.Th s.H.L s.M.Rougier.JA the embrw Feijo, genesis of dicotyledo 97(200010643-l trbution of cyto mic Ca2 and Ca inux ete fu B7 complement 8甲 is of plant develop- s.C.Hagel.H.rz,E.Kra Plant Embryoger si developmental stages and tim gg.zy Am1.Bt.551968)807-81 Faure,Fe 1431 A.AM.Van La [22]N.Huck.J.M.Moore.M.Federe er.U.Grossniklaus,The Ara in p.86(19861-3 utant fen 130203)2149-215y during double fertilization in Zea mys LPlant J.5 23】 Kim der.The tance of being Es EMBRYO 3312008)726 n,E.King.JR.Jord a.The origin and mainte [47 70(0 alysis of the fertil- bidof the GN A ic do Cel [48 ulates a battery of synergid- of GUS arker lines for ger exr a T Ku GENERA or angiosperm fertiliz of maire.S vol.8.Sp 50R DE I Bm22D。na a.Sex.Plant Reprod.12 r.En m:the c oad of seed development.Cun nThe centra [52]LH.Ka BIG.S effen.M.F.Portereiko,A.Lloyd.G.N.Drew 77 tial fertilization i aheeulhte Cell 20(2 ogni n angiosperm 47 [33]H.Ro USA (19) in the path way,Plar t Cell 6 (19 )1241

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