MAIZE EMBRYOGENESIS

ABSTRACT - After a century of morphological descrip-tions and classical genetics maize embryogenesis hasbeen approached over the past decade mainly by molec-ular genetics. Using forward genetics the cloning of adozen mutations causing aberrant embryo developmenthas been accomplished leading to the conclusion thatmutants with developmental blocks before the coleoptilarstage are more likely to be affected in basic cellular func-tions than mutants with later blocks or mutants with vi-able but altered seedlings, which are more likely to beimpaired in regulatory genes. By reverse genetics numer-ous genes with well defined, temporally and/or spatiallyrestricted expression patterns in the maize embryo havebeen isolated and functions inferred based on sequenceanalysis and/or expression patterns. In parallel the phe-notypic analysis of wildtype and mutant embryo mor-phology and cytology has made a step forward by the in-tegration of novel methods such as confocal laser scan-ning microscopy, in situ hybridisation with marker genesor TUNEL assays for the detection of PCD.

KEY WORDS: Embryogenesis; Morphology; Mutant; Mark-er gene; Zea mays.

INTRODUCTION

Embryogenesis is defined as the sum of allgrowth and differentiation processes during the de-velopment of a single-celled zygote into a multi-cel-lular, highly organised mature embryo. The forma-tion of the zygote by the fertilisation of the egg cellby one of the sperm cells marks the transition fromthe gametophytic to the sporophytic generation. Itis part of the double fertilisation event typical offlowering plants that also gives rise to the en-

dosperm by the fertilisation of the central cell bythe second sperm cell (Fig. 1). As in other plantsmaize embryogenesis can be divided into three ma-jor phases. The first one is devoted to developmen-tal events such as pattern formation responsible forthe polarity and position of organs, morphogenesisgiving their shape to organs and differentiation sen-su strictu setting apart epidermis, ground tissue andvascular tissue. The second or maturation phase ismarked by the growth of the embryo and by the ac-cumulation of reserve substances, even thoughsome additional developmental events take place.During the third phase the embryo dehydrates andenters into dormancy to prepare for its separationfrom the mother plant during seed dispersal. Duringgermination the maize embryo becomes theseedling and consumes its own reserve substancesas well as the ones stored in the endosperm thatceases to exist.

While the concepts and the vocabulary of plantembryology are strongly influenced by that of ani-mal systems such as Caenorhabditis, Drosophila ormammals, fundamental differences exist betweenthe two eukaryotic kingdoms. Firstly, plant embryo-genesis is not a distinct process leading to the for-mation of a miniature version of the adult organismcontaining at least primordia of all its future organs.It is rather the beginning of a continuous develop-mental process interrupted temporarily by dorman-cy. While the primary root and some but not all leafprimordia are present in the maize embryo, it doesnot contain primordia of lateral roots, additionalleaves or floral organs. However, the stem cells pre-sent in the shoot apical meristem and root meristemhave the capacity to form secondary meristemswhich in turn give rise to these additional organs(KAPLAN and COOKE, 1997). Secondly, it is generallyrecognised that the elaboration of the body plan isnot based on cell lineage but on the position of in-

Maydica 50 (2005): 469-483

MAIZE EMBRYOGENESIS

V. Vernoud, M. Hajduch, A.-S. Khaled, N. Depège, P.M. Rogowsky*

RDP, UMR 5667 CNRS-INRA-ENSL-UCBL, IFR128 BioSciences Lyon-Gerland,ENS-Lyon, 46 Allée d’Italie, F-69364 Lyon Cedex 07, France

Received January 31, 2005

* For correspondence (fax +33 4 72 72 86 00; [email protected]).

dividual cells within the embryo (JÜRGENS et al.,1994). Instead gradients of hormones or other sig-nalling molecules seem to determine the fate of in-dividual cells (VOGLER and KUHLEMEIER, 2003). Third-ly, the timing and orientation of cell divisions,which are of uttermost importance for embryoshape, are likely governed differently in animalsand plants. Plants have a unique mode of cytokine-sis involving plant-specific structures such as thephragmoplast, a highly dynamic cytoskeletal array,or the preprophase band, a transient structure pre-determining the division plane (JÜRGENS, 2003). Fi-nally the often cited difference between a coenocyt-ic development in animals and a cellular develop-ment at the very beginning of embryo plant devel-opment applies to the Drosophila embryo but notto other animal embryos such as the mouse em-bryo.

RESULTS AND DISCUSSION

Morphology of the maize embryoHistorically the first morphological descriptions

of the maize embryo were side products of work onthe double fertilisation (GUIGNARD, 1901; MILLER,1919; WEATHERWAX, 1919) or on the transformationof the ovary wall into the pericarp of the maturekernel (TRUE, 1893; GUERIN, 1899). Later on morespecialised light microscopical studies were under-taken (AVERY, 1930) and the first comprehensive de-scription and illustration of all developmental stagesof the maize embryo can be found in the timelesswork of Randolph (RANDOLPH, 1936) that remains areference until our days. Another landmark was thestudy by Abbe and Stein (ABBE and STEIN, 1954)who focussed on quantitative aspects including sta-tistically valid cell size measurements or cell num-

470 V. VERNOUD, M. HAJDUCH, A.S. KHALED, N. DEPÈGE, P.M. ROGOWSKY

FIGURE 1 - Embryogenesis in the maize life cycle. Schematic drawing of the life cycle of a maize plant indicating the different phases ofembryo and endosperm development and their duration. DAP, days after pollination.

ber counts throughout embryo development. Withthe advent of electron microscopy in particular theearly stages of maize embryogenesis were revisitedby DIBOLL (1968) and VAN LAMMEREN (1986b), whoprovided additional insight in the spatial and tem-poral aspects of meristem formation. A rather sim-plified schematic summary of this and other work ispresented in Fig. 2. Briefly, the zygote undergoes anasymmetric division into a small apical and a largebasal cell giving rise to the embryo proper and thesuspensor, respectively. The radial symmetry of theproembryo shifts to a bilateral symmetry at the tran-sition stage, which is also characterised by the for-mation of a distinct external cell layer, the proto-derm. At the onset of the coleoptilar stage the shootapical meristem (SAM) and the root apical meristem(RAM) can be distinguished and soon thereafter asmall protuberance marks the position of the futurecoleoptile. The subsequent stages are numberedfrom 1 to 6 according to the number of leaf primor-dia present in the embryo. While the suspensor de-generates, the other parts of the embryo keep grow-ing and reserve substances are accumulated in thescutellum.

Maize has been a model species for the study ofmonocot embryo development and even todaythere are no equally detailed descriptions coveringthe entire embryo development of other grassspecies such as wheat (SMART and O’BRIEN, 1983) orbarley (NORSTOG, 1972; ENGELL, 1989) and in particu-lar rice (MOLDENHAUER and GIBBONS, 2003). In dicotsthe model species for embryo development hasbeen for many years Capsella bursapastoris(HANSTEIN, 1870; SCHULZ and JENSEN, 1968a,b). Inmore recent years it has been marginalised by Ara-bidopsis thaliana for which a reference develop-mental chart had to be established prior to thestudy of numerous embryo mutants (MANSFIELD andBRIARTY, 1991, 1992; MANSFIELD et al., 1991). Similarlyto the maize zygote the Arabidopsis zygote under-goes an asymmetric division giving rise to the em-bryo proper and the suspensor. In the next roundsof cell division the suspensor divides more rapidlythan the embryo proper but ceases to grow soonthereafter. In the embryo proper a protoderm is setaside at the dermatogen stage. After the globularstage SAM and RAM formation is initiated at the be-ginning of the heart stage. A vascular system is dis-tinguishable at the torpedo stage. Embryogenesis iscomplete after accumulation of reserve substancesin the expanding cotyledons (Fig. 2).

The maize and other monocot embryos share

with the Arabidopsis embryo the major functionalprocesses established for the embryogenesis of di-cots (KAPLAN and COOKE, 1997): formation of a zy-gote, establishment of an apical-basal polarity lead-ing to a linear proembryo divided into suspensorand embryo proper, initial histogenesis resulting inthe formation of a protoderm and organisation ofthe two apical meristems at the shoot and root end.On the other hand the morphology of the maizeand Arabidopsis embryo is quite different. A firstdifference concerns the first divisions of the embryothat are rather synchronised and equal in Arabidop-sis leading to easily recognisable geometric figuresand more erratic in maize reflecting possibly the ab-sence of any particular organisation (SHERIDAN,1995). However, the statement needs to be some-how qualified because some maize embryos showrather geometric figures at the 4-cell stage (RAN-DOLPH, 1936) and in Arabidopsis cell numbers differ-ent from 2n can be observed, in particular in thesuspensor (BOWMAN, 1993). Secondly, the formationof leaf primordia occurs after the entrance into dor-mancy and seed dispersal in Arabidopsis, while 5 to6 leaf primordia are elaborated in the maize em-bryo. This difference reinforces the view that thereis no real end to embryogenesis in plants and thatdormancy is a rather arbitrary interruption of a con-tinuous process covering the entire life span of aplant. Thirdly, the axis between SAM and RAM coin-cides with apical-basal axis defined by the suspen-sor and the embryo proper in Arabidopsis but isoblique in maize. This may be the consequence ofthe absence of a second cotyledon. Finally, the rela-tionship between the cotyledons of Arabidopsis andthe scutellum of maize needs to be clarified. Theyare clearly functionally equivalent due to the factthat the vast majority of reserve lipids and proteinsare deposited in these organs. Developmentallyboth arise without involvement of the SAM (JÜRGENS,2001), although this is a matter of debate in Ara-bidopsis (KAPLAN and COOKE, 1997). In maize the de-bate centers around the question whether thescutellum presents all of the cotyledon, part of it ora different structure (KIESSELBACH, 1949). This ques-tion is closely linked to the status of the coleoptilewhich has been interpreted as a new acquisition(BROWN, 1960), the first leaf (GUIGNARD, 1975) orpart of the scutellum (VAN LAMMEREN, 1986b).

Beyond these developmental and phylogeneticconsiderations there are obviously important quanti-tative differences in the size or cell number ofmaize and Arabidopsis embryos, the maize one be-

MAIZE EMBRYOGENESIS 471

ing roughly 10 times larger at maturity. The maizeembryo also takes a 6 times longer period to reachmaturity, the additional time being devoted to thestorage of larger quantities of reserve substancesand to a further progress in the life cycle of theplant with the elaboration of leaf primordia.

A last major difference between maize and Ara-bidopsis concerns the environment of the embryo inthe seed (Fig. 3). In Arabidopsis the endosperm isnonpersistant and the mature seed is essentiallycomposed of the embryo. In maize the endospermpersists and accounts for roughly 2/3 of the maturekernel. Consequently the maize embryo has not on-ly the lipid and protein reserves stored in the scutel-lum but also the carbohydrate reserves stored in theendosperm available during germination.

Cytology of the maize embryoThe light and electron microscopic analysis of

embryo tissue sections was not only a means to re-construct the three dimensional morphology of themaize embryo, it also allowed the observation of cy-tological details. For example the cells of the embryoproper are small and rich in cytoplasm, while thecells of the suspensor are large and highly vacuolat-ed (SCHEL et al., 1984). Epidermal cells are charac-terised by a large nucleus in a central position, ahigh frequency of polysomes, a low number of vac-uoles and the absence of starch granules. The forma-tion of the SAM and RAM involves a dedifferentiationof cells present in the respective positions that mani-fests itself by a decrease in size a loss of vacuolesand an increase of cytoplasm (VAN LAMMEREN, 1986b).


FIGURE 2 - Embryo development in Arabidopsis and maize. Schematic drawing of key developmental stages of Arabidopsis (top) or maizeembryos (bottom). From left to right the stages depicted for Arabidopsis thaliana are zygote, 2-celled, quadrant, dermatogen, globular,heart, torpedo and mature and for Zea mays zygote, 2-celled, early proembryo, late proembryo, transition, early coleoptilar, late coleopti-lar, stage 1 and mature. The time scale in days after pollination (DAP) is merely indicative as embryo development is strongly dependenton the genetic background and environmental conditions. No common scale was used for the drawings to allow detailed views of eachspecies and stage. Only the sizes of the mature embryos are shown.

Cytological studies also give some insight intothe establishment of embryo polarity. A rather dra-matic shift in cell polarity takes place in the egg cellupon fertilisation. While the nucleus and most ofthe cytoplasm are located at the micropylar half ofthe egg cell prior to fertilisation they are found inthe antipodal half afterwards (VAN LAMMEREN, 1986a).More detailed kinetic studies based on the clearingtechnique documented an intermediate positionduring karyogamy that occurs 14 to 18 hours afterpollination. The shift of polarity is completed duringa resting period of 13 to 16 hours before the first di-vision (MOL et al., 1994). After the shift the polarityis the same as in the Arabidopsis or Capsella egg

cell and coincides with the orientation of the futureembryo: embryo proper cells rich in cytoplasm atthe antipodal end and highly vacuolated suspensorcells at the micropylar end. This active reorientationof the cell content is an indication that the polarityof the embryo is not predetermined by the polarityof the egg cell but actively established in the zygoteas a consequence of fertilisation.

Programmed cell death (PCD) is a geneticallyand cytologically well defined auto-destructivemechanism triggered by developmental or environ-mental signals. During development it is a means toeliminate cells that have fulfilled their function andcan be seen as the counterpart of cell division in


FIGURE 3 - Seed development in Arabidopsis and maize. Schematic drawing of key developmental stages of Arabidopsis (top) or maizeseeds (bottom). The time scale in days after pollination (DAP) is merely indicative as seed development is strongly dependent on thegenetic background and environmental conditions. No common scale was used for the drawings to allow detailed views of each speciesand stage.

the morphogenesis of organs. The underlyingmechanisms of PCD in plants are less well knownthan those of PCD in animals, but several morpho-logical and biochemical similarities exist such asDNA laddering, caspase-like proteolytic activity orcytochrome C release from mitochondria (HOE-BERICHTS and WOLTERING, 2003). Based on the TUNELmethod allowing the visualisation of DNA fragmen-tation on cytological sections, PCD was detected inthe suspensor, scutellum, coleoptile and root cap ofthe developing maize embryo (GIULIANI et al., 2002).At stage 1 the main activity concerns the suspensorwhere the PCD progresses from the top to the bot-tom during further developmental stages. Consider-able PCD is also observed in the scutellum with agradient from the adaxial to the abaxial side, whilethe TUNEL staining in the coleoptile and root cap isless intense (Fig. 4). These experiments suggest thatthe “degeneration” of the suspensor that had beendescribed for decades is not a simple necrosis but adevelopmentally regulated process. Similarly the de-position of reserve substances in the scutellum isfollowed by a coordinated disassembly of the corre-sponding cells. None of the TUNEL positive parts ofthe embryo contribute to the adult plant body lead-ing to the hypothesis that PCD may be the means toeliminate purely embryonic structures as soon asthey are no longer needed.

In vitro culture of the maize embryoSince the young maize embryo is deeply buried

in maternal tissues (Fig. 3), it is not readily accessi-ble for experimentation. Consequently a lot of efforthas been invested in the in vitro culture of maizeembryos that allows the application of hormones orother signal molecules and facilitates the microscopi-cal observation. In addition to androgenesis (PETOLI-NO and JONES, 1986), gynogenesis (BECKERT, 1994)and somatic embryogenesis (BAUDINO et al., 2001)the culture of zygotic embryos of various stages hasbeen achieved (MATTHYS-ROCHON et al., 1998). Em-bryos excised before the transition stage depend fortheir development on the presence of cytokinins inculture medium, while older embryos develop intomature and fertile plants without any external hor-mone supply. Embryo culture has been used for thecharacterisation of mutant embryos allowing conclu-sions as to the type of the lesion depending on theoutcome of the rescue experiment (SHERIDAN andNEUFFER, 1980; CONSONNI et al., 2003). In wheat theapplication of auxin polar transport inhibitorsdemonstrated that auxin has a determining influence

on the differentiation of the embryonic axis and thescutellum in grass embryos (FISCHER et al., 1997).

The culture of very young embryos before theproembryo stage is hampered by the difficulties ofproper micro-dissection without surrounding tis-sues. Two experimental solutions exist: either thezygote or 2-celled embryo is excised with the sur-rounding endosperm and some nucellar tissue (MOL

et al., 1993) or clean zygotes are obtained by treat-ment with a mix of cell wall degrading enzymes(LEDUC et al., 1996). In both cases only a certain per-centage of the cultured zygotes follows a normaldevelopment and yields fertile plants. Contrary tothe zygotes cultured in the natural environment ofthe embryo sac and some nucellar tissue, the cleanzygotes require the co-culture with androgeneticmicrospores for their development. In addition theirdevelopment is not direct but occurs via a callusphase and secondary embryogenesis after reachingthe transition stage. This data suggest that the zy-gote and the very young embryo need external sig-nals for proper development.

In vitro culture has also been used to study thedynamics of the fertilisation process. Isolated eggcells and sperm cells can be fused in vitro and fer-tile plants be generated via secondary embryogene-sis (KRANZ and LÖRZ, 1993; FAURE et al., 1994). Afteran adhesion of several minutes the gametes fuse inless than 10 s. This process is rather specific tomale-female gamete pairs and establishes a barrierto polyspermy. Further experiments with fluores-


FIGURE 4 - Programmed cell death in the maize embryo. In aschematic drawing of a longitudinal (left) or transverse section(right) of a stage 1 maize embryo zones of intense TUNEL stain-ing are indicated in dark red, zones of weak staining in pink andTUNEL negative zones in white. The dashed line in the longitudi-nal section indicates the plane of the transverse section.

cent Ca2+ indicator dyes showed that the cytoplas-mic fusion triggers a transient increase in cytosolicCa2+ in the fertilised egg cell that lasts several min-utes (DIGONNET et al., 1997). The Ca2+ is of extracel-lular origin and enters the cell at the vicinity of thesperm entry site approximately 2 s after fusion. Sub-sequently the Ca2+ entry is gradually generalisedover the entire zygote membrane (ANTOINE et al.,2000). One may speculate that the initial calciumgradient or the subsequent elevated cytosolic calci-um level is a coordinator in space and time of de-velopmental events in the zygote such as the inver-sion of polarity or the activation of transcription.

Mutants of the maize embryoEmbryo mutants have been isolated and studied

since the beginning of the last century (DEMERC,1923). As other maize mutants they were obtainedeither by EMS mutagenesis or from stocks with ahigh frequency of transposition. These stocks con-tain active transposable elements, generally of theMutator (Mu), Activator (Ac) or Enhancer (En)class, that transpose either themselves or mediatethe transposition of truncated elements of the sameclass. From the very beginning the nomenclature ofmutants with smaller, incompletely developed oraberrant embryos has caused some problems due tooverlaps in the definition of mutant classes, for ex-ample between defective seed (de) (JONES, 1920;MANGELSDORF, 1923) and germless (gm) mutants (DE-MERC, 1923). Both are part of the large class of de-fective kernel (dek) mutants that include in the origi-nal definition by NEUFFER and SHERIDAN (1980) muta-tions affecting either the endosperm or the embryoor both of them and where in the last two cases themutant embryo is either not viable or develops intoa seedling with a distinct mutant phenotype (NEUF-FER and SHERIDAN, 1980). Work with this class of ker-nel mutants is hampered by the fact that the vastmajority are single gene recessive mutants that arenot viable in the homozygous form and need to bepropagated as heterozygotes. Viable kernels are al-ways a mixture of wildtype and heterozygote ker-nels and, in the absence of molecular markers, theresulting plants can only be distinguished a posteri-ori by scoring self-pollinated ears. Similarly, at earlydevelopmental stages homozygous mutant kernelsare often not distinguishable from wildtype or het-erozygous kernels by simple visual inspection andonly a more detailed analysis such as the produc-tion of cytological sections of randomly sampledkernels allows the identification and observation of

mutant embryos. As a consequence there was a hia-tus of 50 years in the work on dek mutants and veryoften the analysis of the mutant phenotype was re-stricted to observations at kernel maturity.

The modern era of dek mutant characterisationstarts with the EMS induced collection of NEUFFER

and SHERIDAN (1980) consisting of 855 recessive ker-nel mutants. Genetic, lethality, morphological, andembryo rescue studies on nearly 200 dek mutantsshowed (1) that dek mutations can occur on at least17 of the 20 chromosome arms, (2) that the pres-ence of a normal endosperm can have a positive,neutral or negative effect on a mutant embryo, (3)that the embryo is generally more severely defectivethan the endosperm, (4) that the majority of em-bryos is blocked at or after stage 1, and (5) thatmore than one half of the mutant embryos can berescued by tissue culture (NEUFFER and SHERIDAN,1980; SHERIDAN and NEUFFER, 1980). A more detailedstudy of 14 mutants with defects in both embryoand endosperm at early developmental stages re-vealed that the embryo phenotype manifests itselfeven in the presence of wildtype endosperm andthat in some instances mutant endosperm can im-pair the germination of wildtype embryos. These re-sults obtained by pollination of heterozygotes withB-A translocation stock suggest that the embryophenotype is not simply a consequence of insuffi-cient nutrient supply by mutant endosperm and thatmutant endosperm may contain toxic substances(SHERIDAN and NEUFFER, 1982). For 7 of these mu-tants detailed developmental profiles of the mutantembryo were established by series of cytologicalsections throughout the development (CLARK andSHERIDAN, 1986; SHERIDAN and THORSTENSON, 1986;CLARK and SHERIDAN, 1988). A great variability of thephenotype was observed because mutant embryoscan be arrested at the proembryo, transition orcoleoptilar stage, can be necrotic or not, and cansimply stop growth or proliferate without everforming leaf primordia.

Since positional cloning of EMS mutants wasconsidered as unfeasible in maize, interest shifted totransposon-tagged embryo mutants. A collection of63 dek mutants obtained after a cross with activeMutator stock was well characterised genetically bychromosome arm locations for 53 mutants and bylinkage mapping and allelism tests for 21 mutants(SCANLON et al., 1994). While the initial phenotypiccharacterisation concerned only the gross morphol-ogy of the kernel at maturity, the detailed pheno-types of individual mutants were the subject of sub-


sequent work. The viable embryos of the pleiotrop-ic semaphore1 (sem1) mutant are smaller than wild-type embryos and have fewer if any leaf primordia.Ectopic Knox gene expression in leaves and en-dosperm and reduced polar auxin transport suggestthat Sem1 may be an intermediate in a signallingcascade leading from auxin to Knox genes that inturn are necessary for the correct initiation of leafprimordia (SCANLON et al., 2002). The discoloured-1(dsc1) mutation results in an arrest of embryogrowth at stage 1 followed by tissue degradation.Part of the mutated gene has been cloned but nofunction has been deduced from the partial se-quence (SCANLON and MYERS, 1998). Embryos of theempty pericarp2 (emp2) mutant reach an abnormalcoleoptilar stage characterised by a SAM lacking thetypical tunica-corpus shape and incapable of form-ing leaf primordia (SCANLON et al., 1997). The under-lying gene shows significant homology to animalheat shock binding proteins and encodes a negativeregulator of heat shock response in maize (FU et al.,2002). The embryo lethal phenotype suggests that,in addition to their protective role during heatstress, heat shock proteins (HSP) may have a devel-opmental role in plants as it has been documentedin animals (CHRISTIANS et al., 2003). The molecularcloning of the latter two dek mutations wasachieved via Mutator tags and confirmed the initialhypothesis that at least some of the newly arisingmutants after a cross with active Mutator stock werecaused by the insertion of one of the 9 Mutator ele-ments (SCANLON et al., 1994).

Several other dek mutants have been charac-terised, although the phenotypic descriptions fre-quently focused on the endosperm rather than theembryo. For two of them the underlying genes havebeen identified via a Mutator and an Activator tag,respectively. Dek1 encodes a membrane proteinwith a cytoplasmic cysteine proteinase domain at itsC-terminus. The exact role of this protein in the de-velopment beyond the transition stage and the es-tablishment of the embryo axis remains to be deter-mined (BECRAFT et al., 2002; LID et al., 2002). Lachri-ma (DekB) encodes a transmembrane protein ex-pressed very early in kernel development with apreference for proliferating tissues of the embryo.Mutant embryos are blocked at the transition stageand undergo little change until kernel maturity.Based on structural rather than sequence similaritieswith Arabidopsis proteins Lachrima may be an aux-in transporter, which would readily explain the em-bryo phenotype (STIEFEL et al., 1999).

In an attempt to focus on developmental ratherthan metabolic defects in embryo development, aMutator derived collection was screened for em-bryo-specific (emb) mutants (CLARK and SHERIDAN,1991). Considered by some but not all authors as asub-class of dek mutants, emb mutants are definedby defects concerning only the embryo but not theendosperm. Also called germless or lethal embryo,these mutants are more likely to be affected in de-velopmental processes because metabolic defectsshould also manifest themselves in the endospermthat shares with the embryo a heterotrophic growth.The embryo morphology of 51 independent mu-tants was documented at kernel maturity andshowed that frequently the embryos not simplystopped growth but were morphologically abnor-mal, that in about 2/3 of the mutants, the embryowas arrested prior to stage 1 and that embryonecrosis was found only in very few mutants (CLARK

and SHERIDAN, 1991; SHERIDAN and CLARK, 1993). Acytological analysis of 5 non-allelic mutants blockedbefore the transition stage in the course of embryodevelopment revealed three phenotypic groups:morphologically normal embryos that ceasedgrowth at the transition stage, tube-shaped embryoslacking apical-basal differentiation and embryoswith a proliferation of the suspensor tissue (HECKEL

et al., 1999). The gene responsible for the latterphenotype in mutant emb*-8516 was identified asZmPRPL35-1 encoding a plastid ribosomal protein(MAGNARD et al., 2004). Interestingly, a lesion in an-other nuclear-encoded plastid ribosomal protein,ZmPRPS9, also affects only the embryo and not theendosperm in the lem1 mutant (MA and DOONER,2004). It is not clear at this point in time whetherthe embryo is more dependent on functional plas-tids than the endosperm or whether the two pro-teins have extra-ribosomal functions required forembryogenesis.

From the same collection a second group of fouremb mutants with slightly later developmentalblocks was characterised by confocal laser scanningmicroscopy. Two of them ceased growth at theproembryo stage. Based on protoderm marker geneexpression the other two underwent an incompleteradial organisation that compromised without com-pletely abolishing the formation of a SAM or leafprimordia (ELSTER et al., 2000). The major conclusionfrom these data is that protoderm and meristem for-mation are not independent events but that proto-derm formation is a prerequisite for proper meris-tem formation.


The characterisation of another group of threeindependently isolated non-allelic emb mutants witha developmental block prior to the transition stageshowed abnormal proliferation of the suspensorand absence of PCD in both the suspensor and thescutellum (CONSONNI et al., 2003). Surprisingly em-bryo rescue on hormone-free medium with sucrosegives rise to small but otherwise normal seedlingssuggesting that the culture conditions trigger theformation of functional meristems. These data fur-ther support the observation that the suppression ofmorphogenesis is frequently accompanied by anuncontrolled pattern of cell division.

Other sub-classes of dek mutants can providevaluable information on embryo development, andin particular defective seedling (des) mutants. Em-bryos of this sub-class germinate but develop intoaberrant seedlings that do not yield mature plants.The seedling defects can generally be traced backto aberrations already detectable before germina-tion. In Arabidopsis the screen for seedling ratherthan embryo defects has been established early onas the method of choice for the isolation of patternmutants (MAYER et al., 1991). The most prominentexample in maize is the shootless phenotype, inwhich the embryo possesses a scutellum and theroot half of the embryo axis but completely lacksthe shoot half of the embryo axis. This intriguingphenotype manifests itself only in double mutantsat the shootmeristemless (sml) and distorted growth(dgl) loci. Mutant seedlings form an apparently nor-mal primary root but never develop abovegroundorgans. The in vitro culture of mutants on a medi-um with high levels of cytokinin leads to the con-clusion that the double mutant may be affected inthe perception of cytokinin signals (PILU et al.,2002).

The class of viviparous (vp) mutants is definedby a precocious germination of the embryo on theear. These mutants are a good illustration of theconcept that desiccation and dormancy are a ratherarbitrary endpoint of embryogenesis and that thereis a continuity between embryonic and post-embry-onic development. While some of the resultingseedlings are lethal, others produce normal fertileplants. Biochemical analyses showed that several vpmutations are affected in carotenoid and ABA syn-thesis (NEILL et al., 1986). This is the case for vp5which has been cloned and encodes a phytoene de-saturase (LI et al., 1996) but not for the well knownvp1 encoding a novel type of plant-specific tran-scription factors (MCCARTY et al., 1991). A role of the

plant hormone ABA in the desiccation process ofthe kernel and in particular of its balance with GAin the control of germination is well documented(WHITE et al., 2000).

Since maize embryos contain up to 6 leaf pri-mordia, many mutants disturbed in the organisationof the SAM and/or leaf morphology after germina-tion are also relevant for embryogenesis. Most ofthe phenotypic analysis of these mutants has fo-cused on post-embyronic stages and will not be dis-cussed here. Interestingly the genes responsible ofthe phenotypes in Knotted1 (Kn1) (VOLLBRECHT etal., 1991), Rough sheath1 (Rs1) (SCHNEEBERGER et al.,1995), rough sheath2 (rs2) (TIMMERMANS et al., 1999),Gnarley1 (Gn1) (FOSTER et al., 1999), narrowsheath1 and 2 (ns1, ns2) (NARDMANN et al., 2004) orterminal ear1 (te1) (VEIT et al., 1998) all code forputative transcription factors. Transcriptional regula-tion plays a major role in animal embryogenesisand consequently the regulatory gene functions al-tered in meristem mutants are more readily recon-cilable with a developmental role than the cellularfunctions impaired in dek or emb mutants.

Genes of the maize embryoMaize is an ancient allo-tetraploid and duplica-

tions of large parts of the genome are well docu-mented (GAUT and DOEBLEY, 1997). Consequentlythe analysis of single gene recessive mutants is like-ly to uncover only part of the genes involved in agiven developmental process. Gene expression andin particular differential gene expression is an alter-native criterion to identify additional candidategenes even though their implication in the processneeds to be confirmed by the characterisation ofmutants or transgenic plants. The most comprehen-sive analysis to date was based on a thematic arraycontaining 900 selected genes and 600 randomcDNA clones from a 20 DAP embryo library. Its hy-bridisation with kernel or embryo probes obtainedbetween 5 and 45 DAP established several charac-teristic temporal expression profiles in the embryothat can be correlated with gene function anddemonstrated that co-expression was stronger forgenes involved in the TCA cycle rather than in gly-colysis (LEE et al., 2002). On the protein level recentdata with spot identification by mass spectrometryare available for the endosperm (MECHIN et al.,2004), while only profiles of non-identified spotsexist for the embryo (SANCHEZ-MARTINEZ et al., 1986).

Since it is impossible to review all genes forwhich expression in the embryo has been demon-


strated, we will focus on genes that present either aspecific or preferential expression in the embryo ascompared to other parts of the maize plants orgenes whose expression is restricted to particularparts or developmental stages of the embryo. Geneactivation after fertilisation was addressed by a dif-ferential screen between an egg cell and a zygotecDNA library. Only 2% of the 4000 surveyed clonesexhibited significant changes in transcript levels.Among them was a calreticulin that could possiblyplay a role in the calcium wave observed in the zy-gote after fertilisation (DRESSELHAUS et al., 1996). Inaddition a global silencing of the paternal genomeduring early embryogenesis can be excluded be-cause a Gfp transgene provided by the male parentis transcribed as early as 4 h after fertilisation(SCHOLTEN et al., 2002). Global paternal silencing hasbeen reported in Arabidopsis but remains a contro-versial issue (VIELLE-CALZADA et al., 2000; WEIJERS etal., 2001).

Genes belonging to the Outer cell layer (OCL)family may play a role in protoderm formation andmaintenance because their expression is restrictedto the outermost cell layer of the embryo prior tothe cytologically visible differentiation of the proto-derm at the transition stage (INGRAM et al., 1999).Genes OCL1 to 5 encode putative transcription fac-tors of the HD-ZIP IV family and their overlappingbut distinct expression patterns in different parts ofthe protoderm suggest that the protoderm is not auniform entity but divided in developmental territo-ries (INGRAM et al., 2000). Similarly the Lipid transferprotein2 (Ltp2) gene is expressed only in the abaxi-al protoderm of the scutellum and coleoptile (SOS-SOUNTZOV et al., 1991).

Meristem formation in the embryo is accompa-nied by the onset of Kn1 expression in the corre-sponding regions. Restricted to the SAM region attransition stage expression spreads all the way tothe RAM at stage 1 but remains excluded from theincipient leaf, leaf primordia and the scutellum(SMITH et al., 1995). Loss-of-function mutants of Kn1have severe inflorescence and floral phenotypes butstill seem to have a functional SAM (KERSTETTER etal., 1997). On the other hand the complete loss ofthe putative orthologue Shootmeristemless in Ara-bidopsis causes failure to develop a SAM during em-bryogenesis (LONG et al., 1996). These results sug-gest a redundant function in maize and are reminis-cent of the uncloned sml mutant in which two locineed to be mutated to uncover the phenotype (PILU

et al., 2002). While detailed analyses document the

expression of other members of the Knotted-likehomeobox (Knox) family in various part of the veg-etative shoot apex, their expression pattern has nev-er been rigorously established in embryos. RNA gelblots prove expression of Knox2, Knox4/Gn1 andKnox6 in 17 DAP embryos but fail to detect the ex-pression of other Knox genes including Kn1, whichis known to be expressed by in situ hybridisation(KERSTETTER et al., 1994). An antibody recognisingseveral KNOX proteins marks a territory similar tothe one described for Kn1 (SCANLON et al., 2002).Other than Knox genes, Ns1 and Ns2 are involvedin the elaboration of lateral organs from the SAM.Their expression is first detected in coleoptilar em-bryos where they mark the lateral margins of theemerging coleoptile. The post-embryonic expres-sion patterns of these putative transcription factorsof the Wuschel family are also highly dynamic andalways restricted to small groups of cells at tissueboundaries (NARDMANN et al., 2004).

For several other genes detailed developmentalexpression profiles have been established withoutshedding much light on their precise function.Among the Fertilisation-independent endosperm(Fie) genes the kernel-specific Fie1 is not expressedin the embryo, while the constitutively expressedFie2 was found in the embryo proper at the proem-bryo stage and in leaf primordia at later stages(DANILEVSKAYA et al., 2003). As their Arabidopsiscounterparts they are likely involved in chromatinremodelling (LUO et al., 2000). Four ZmHox genesare co-expressed in the embryo proper at theproembryo stage and in the embryo axis at laterstages. They encode putative transcription factors ofthe PHD subclass of homeodomain proteins (KLINGE

and WERR, 1995). While ZmHox1 has been isolatedas a transcriptional regulator of Shrunken1 encod-ing sucrose synthase in the endosperm, its role inthe embryo is not clear (BELLMANN and WERR, 1992).Hydroxyproline-rich glycoprotein (HPRP) is ex-pressed mainly in the vascular system of the em-bryo axis and may intervene in early steps of cellwall synthesis (RUIZ-AVILA et al., 1992). Hybrid pro-line-rich protein (HyPRP) shows a complementaryexpression pattern with little expression in the em-bryo axis and a gradient-like expression in thescutellum (JOSE-ESTANYOL et al., 1992). Expression isstrongest in cells not yet reached by PCD.

During the maturation phase two types of pro-teins have attracted attention. Oleosins are small hy-drophobic proteins localised in the phospholipidmono-layer that constitutes the envelope of oil bod-


ies. Oil bodies are organelles filled with fatty acidsthat present the major reserve substance stored inthe embryo. Three Oleosin (Ole) genes have beencharacterised that are co-ordinately regulated duringkernel maturation and that can be found both in theembryo axis and in the scutellum (LEE and HUANG,1994). Globulins are the major storage proteins ofthe embryo and the proteins encoded by Globulin1and 2 (Glb1, Glb2) account for 10 to 20% of theembryo protein (KRIZ, 1989).

A group of structurally related proteins collec-tively called late embryogenesis abundant proteins(LEA) accumulate during late stages of embryogene-sis and have been associated with dehydration. Asub-group are the structurally related dehydrins(DHN) that can also be found in vegetative tissuesupon water or osmotic stress (CLOSE, 1997) and thatpossibly act as intracellular sponges (GARAY-ARROYO

et al., 2000). Some of the corresponding genes havealso been called Responsive to ABA (Rab) due totheir induction upon treatment with external ABA inboth embryos and vegetative tissues. However, atleast in the case of Rab28 different trans-acting fac-tors are involved in gene induction during embryo-genesis or by ABA treatment (PLA et al., 1993). BothRab17 (=Dhn1) and Rab28 are more strongly ex-pressed in the embryo axis as compared to thescutellum (GODAY et al., 1994; NIOGRET et al., 1996),while no expression data are available for Dhn2(CAMPBELL et al., 1998). On a sub-cellular level nu-clear or cytoplasmic localisation of Rab17 is stronglyinfluenced by phosphorylation via protein kinaseCK2 (GODAY et al., 1994). This phosphorylation is al-so essential for the action of Rab17 during seed ger-mination in transgenic Arabidopsis (RIERA et al.,2004). Similar functional approaches are now need-ed to confirm gene functions of Rab and other ex-pressed genes that have been suggested by se-quence and/or expression data.

CONCLUSION AND PERSPECTIVES

After a century of morphological descriptionsand classical genetics maize embryogenesis hasbeen approached over the past decade mainly bymolecular genetics. Over a dozen mutations causingaberrant embryo development have been cloned.Detailed phenotypic characterisations of these andmany more mutants integrated novel techniquessuch as confocal laser scanning microscopy, in situhybridisation with marker genes or the production

of transgenic plants in maize or other species. As ageneral conclusion mutants with developmentalblocks before the coleoptilar stage are more likelyto be affected in basic cellular functions, while mu-tants with later blocks or mutants with viable but al-tered seedlings are more likely to be impaired inregulatory genes.

The success of the transposon tagging approachin forward genetics of embryogenesis is difficult toassess. While successful cloning of dek or emb mu-tations has been reported for several mutant popu-lations generated from crosses with active Mutatorstock, we and others have experienced a lot of frus-tration with this approach. This may be explainedby the technical difficulties to visualise all Mutatorelements present in a genome or by an increased“spontaneous” mutation rate in active Mutatorstock. One may hypothesise that in active Mutatorstock not only the transposition frequency of Muta-tor elements is increased but also that of numerousother transposable elements (that are not detectedby the Mutator specific methods). The creation ofmore sophisticated populations and/or the conjunc-tion of systematic phenotypic characterisation withsystematic sequencing of flanking sequences willcertainly improve the situation.

While the detailed phenotypic and molecularanalysis of dek or emb mutants provided importantinsights into the mechanisms and genes involved inmaize embryogenesis, several limitations also be-came evident. Firstly, for the majority of the emband other lethal dek mutants the developmentalblocks concern very early stages. Often the mor-phological analysis only allows to determine theprecise stage at which mutant development devi-ates from wildtype development but not to drawany further conclusions. While viable dek mutantsor leaf mutants are generally more informative, sofar none of them concern the first two fundamentalsteps of apical-basal polarisation or protoderm for-mation. Secondly, none of the underlying genescloned so far carry annotations suggesting develop-mental functions. At first sight they are house keep-ing genes involved in basic cellular functions thatare not restricted to embryogenesis. While this wasmore or less expected for dek mutants in general,this was more surprising in the case of emb mu-tants. The challenge in the later case is now to findout why the embryo and the endosperm are not af-fected in the same way, for example by mutationsin genes that are part of the translational machineryof plastids. In this particular case one may specu-


late that embryo but not endosperm developmentmay be dependent on the action of plant hormonessuch as GA or ABA that are partly synthesised inplastids. Finally the embryo lethal phenotypemakes it difficult to asses the role of mutated genesin post-embryonic development. To determinemore precisely the function of these genes it will benecessary to obtain conditional knock-outs that al-low normal growth and development during em-bryogenesis and inhibit expression after germina-tion. This may be accomplished by RNAi constructsunder the control of tissue-specific or induciblepromoters or by clonal mosaic analysis induced byX-ray treatment of the seed as demonstrated for theemp2 mutant (FU and SCANLON, 2004).

Reverse genetics also made valuable contribu-tions to the understanding of embryogenesis. Nu-merous genes expressed in the embryo have beenisolated and characterised and in many cases the se-quence and/or expression pattern give more or lessprecise hints as to their function. Two major pitfallsof this approach are nicely illustrated by the dupli-cate genes Ns1 and Ns2. Firstly, their expression ter-ritory is not only spatially limited to a handful ofcells but also very dynamic in time and can easilybe missed or missed in part by a low-resolutionanalysis. Secondly, single mutants do not show aphenotype due to functional redundancy betweenthe two genes and double mutants need to be con-structed to draw conclusions as to gene function.The bottle neck of reverse genetics is not the isola-tion or molecular characterisation of (differentially)expressed genes but the isolation of correspondingmutants or the production of transgenic plants toconfirm the suspected function, for example of OCLgenes in protoderm formation or of non-clonedKnox genes in meristem formation and mainte-nance. The establishment of public transformationfacilities and the availability of a TILLING popula-tion in addition to several transposon-based popula-tions will certainly speed up this process.

Several of the genes with well defined, tempo-rally and/or spatially restricted expression patternsin the maize embryo can and have been used asmarker genes (BOMMERT and WERR, 2001). Markergenes are a widely used tool in developmental bi-ology that allow to identify morphologically aber-rant tissues or to establish hierarchical links be-tween genes. The characterisation of the precisetemporal and spatial expression patterns of addi-tional genes and gene families in the maize embryowill not only provide additional candidate genes

for functional validation but also a denser map ofmarker genes useful for the in depth analysis of ex-isting mutants.

ACKNOWLEDGMENTS - MH was supported by Marie Curie Fel-lowship QLK1-CT-2001-51051 of the European Commission andASK by a PhD grant of the Egyptian Government.

REFERENCES

ABBE E.C., O.L. STEIN, 1954 The growth of the shoot apex inmaize: embryogeny. Am. J. Bot. 41: 173-284.

ANTOINE A.F., J.E. FAURE, S. CORDEIRO, C. DUMAS, M. ROUGIER, J.A.FEIJO, 2000 A calcium influx is triggered and propagates inthe zygote as a wavefront during in vitro fertilization of flow-ering plants. Proc. Natl. Acad. Sci. USA 97: 10643-10648.

AVERY G.S., 1930 Comparative anatomy and morphology of em-bryos and seedlings of maize, oats and wheat. Bot. Gaz. 89:1-39.

BAUDINO S., S. HANSEN, R. BRETTSCHNEIDER, V.F.G. HECHT, T. DRES-SELHAUS, H. LÖRZ, C. DUMAS, P.M. ROGOWSKY, 2001 Molecularcharacterisation of two novel maize LRR receptor-like kinas-es, which belong to the SERK gene family. Planta 213: 1-10.

BECKERT M., 1994 Advantages and disadvantages of in vitro/insitu produced DH maize plants. Biotechnol. Agricult. For. 25:201-213.

BECRAFT P.W., K.J. LI, N. DEY, Y. ASUNCION CRABB, 2002 Themaize dek1 gene functions in embryonic pattern formationand cell fate specification. Development 129: 5217-5225.

BELLMANN R., W. WERR, 1992 Zmhox1a, the product of a novelmaize homeobox gene, interacts with the Shrunken 26 bpfeedback control element. EMBO J. 11: 3367-3374.

BOMMERT P., W. WERR, 2001 Gene expression patterns in themaize caryopsis: clues to decisions in embryo and en-dosperm development. Gene 271: 131-142.

BOWMAN J., 1993 Embryogenesis. pp. 351-401. In: J. Bowman(Ed.), Arabidopsis: An atlas of morphology and development.Springer Verlag, New York Berlin Heidelberg.

BROWN W.V., 1960 The morphology of the grass embryo. Phyto-morphology 10: 215-223.

CAMPBELL S.A., D.E. CRONE, T.L. CECCARDI, T.J. CLOSE, 1998 A ca.40 kDa maize (Zea mays L.) embryo dehydrin is encoded bythe dhn2 locus on chromosome 9. Plant Mol. Biol. 38: 417-423.

CHRISTIANS E.S., Q. ZHOU, J. RENARD, I.J. BENJAMIN, 2003 Heatshock proteins in mammalian development. Semin. Cell.Dev. Biol. 14: 283-290.

CLARK J.K., W.F. SHERIDAN, 1986 Developmental profiles of themaize embryo-lethal mutants dek22 and dek23. J. Hered. 77:83-92.

CLARK J.K., W.F. SHERIDAN, 1988 Characterization of the two em-bryo-lethal defective kernel mutants rgh*-1210 and fl*-1253B: Effects on embryo and gametophyte development.Genetics 120: 279-290.


CLARK J.K., W.F. SHERIDAN, 1991 Isolation and characterisation of51 embryo-specific mutations of maize. Plant Cell 3: 935-951.

CLOSE T.J., 1997 Dehydrins: a commonalty in the response ofplants in dehydration and low temperature. Physiol. Plant.100: 291-296.

CONSONNI G., C. ASPESI, A. BARBANTE, S. DOLFINI, C. GIULIANI, A.GIULINI, S. HANSEN, R. BRETTSCHNEIDER, R. PILU, G. GAVAZZI,2003 Analysis of four maize mutants arrested in early em-bryogenesis reveals an irregular pattern of cell division. SexPlant Reprod. 15: 281-290.

DANILEVSKAYA O.N., P. HERMON, S. HANTKE, M.G. MUSZYNSKI, K. KOL-LIPARA, E.V. ANANIEV, 2003 Duplicated fie genes in maize:Expression pattern and imprinting suggest distinct functions.Plant Cell 15: 425-438.

DEMERC M., 1923 Heritable characters of maize. XV Germlessseed. J. Hered. 114: 297-300.

DIBOLL A.G., 1968 Fine structural development of the megaga-metophyte of Zea mays following fertilization. Am. J. Bot.55: 787-806.

DIGONNET C., D. ALDON, N. LEDUC, C. DUMAS, M. ROUGIER, 1997First evidence of a calcium transient in flowering plants atfertilization. Development 124: 2867-2874.

DRESSELHAUS T., C. HAGEL, H. LORZ, E. KRANZ, 1996 Isolation of afull-length cDNA encoding calreticulin from a PCR library ofin vitro zygotes of maize. Plant Mol. Biol. 31: 23-34.

ELSTER R., P. BOMMERT, W.F. SHERIDAN, W. WERR, 2000 Analysis offour embryo-specific mutants in Zea mays reveals that in-complete radial organisation of the proembryo interfers withsubsequent development. Dev. Genes Evol. 210: 300-310.

ENGELL K., 1989 Embryology of barley: Time course and analy-sis of controlled fertilization and early embryo formationbased on serial sections. Nord. J. Bot. 9: 265-280.

FAURE J.E., C. DIGONNET, C. DUMAS, 1994 An in vitro system foradhesion and fusion of maize gametes. Science 263: 1598-1600.

FISCHER C., V. SPETH, S. FLEIG-EBERENZ, G. NEUHAUS, 1997 Induc-tion of Zygotic Polyembryos in Wheat: Influence of AuxinPolar Transport. Plant Cell 9: 1767-1780.

FOSTER T., J. YAMAGUCHI, B.C. WONG, B. VEIT, S. HAKE, 1999Gnarley1 is a dominant mutation in the knox4 homeoboxgene affecting cell shape and identity. Plant Cell 11: 1239-1252.

FU S., R. MEELEY, M.J. SCANLON, 2002 Empty pericarp2 encodes anegative regulator of the heat shock response and is re-quired for maize embryogenesis. Plant Cell 14: 3119-3132.

FU S., M.J. SCANLON, 2004 Clonal mosaic analysis of EMPTYPERICARP2 reveals nonredundant functions of the duplicatedHEAT SHOCK FACTOR BINDING PROTEINs during maizeshoot development. Genetics 167: 1381-1394.

GARAY-ARROYO A., J.M. COLMENERO-FLORES, A. GARCIARRUBIO, A.A.COVARRUBIAS, 2000 Highly hydrophilic proteins in prokary-otes and eukaryotes are common during conditions of waterdeficit. J. Biol. Chem. 275: 5668-5674.

GAUT B.S., J.F. DOEBLEY, 1997 DNA sequence evidence for thesegmental allotetraploid origin of maize. Proc. Natl. Acad.Sci. USA 94: 6809-6814.

GIULIANI C., G. CONSONNI, G. GAVAZZI, M. COLOMBO, S. DOLFINI,2002 Programmed cell death during embryogenesis inmaize. Ann. Bot. 90: 287-292.

GODAY A., A.B. JENSEN, F.A. CULIANEZMACIA, M.M. ALBA, M.FIGUERAS, J. SERRATOSA, M. TORRENT, M. PAGES, 1994 Themaize abscisic acid-responsive protein Rab17 is located inthe nucleus and interacts with nuclear-localization signals.Plant Cell 6: 351-360.

GUERIN P., 1899 Recherches sur le développement du tégumentséminal et du péricarpe des graminées. Ann. Sci. Nat. Bot. 9:1-59.

GUIGNARD J.L., 1975 Du cotyledon des monocotyledons. Phyto-morphology 25: 193-200.

GUIGNARD L., 1901 La double fécondation dans le maïs. J. Bot.15: 37-50.

HANSTEIN J., 1870 Die Entwicklung des Keimes der Monokotylenund Dikotylen. Bot. Abh. 1: 1-112.

HECKEL T., K. WERNER, W.F. SHERIDAN, C. DUMAS, P.M. ROGOWSKY,1999 Novel phenotypes and developmental arrest in earlyembryo specific (emb) mutations of maize. Planta 210: 1-8.

HOEBERICHTS F.A., E.J. WOLTERING, 2003 Multiple mediators ofplant programmed cell death: interplay of conserved celldeath mechanisms and plant-specific regulators. Bioessays25: 47-57.

INGRAM G.C., J.L. MAGNARD, P. VERGNE, C. DUMAS, P.M. ROGOWSKY,1999 ZmOCL1, an HDGL2 family homeobox gene, is ex-pressed in the outer cell layer throughout maize develop-ment. Plant Mol. Biol. 40: 343-354.

INGRAM G.C., C. BOISNARD-LORIG, F. DEGUERRY, C. DUMAS, P.M. RO-GOWSKY, 2000 Expression patterns of genes encoding HD-ZipIV homeodomain proteins define specific domains in themaize embryos and meristems. Plant J. 22: 401-414.

JONES D.F., 1920 Heritable characters of maize. IV A lethal fac-tor-defective seeds. J. Hered. 11: 161-167.

JOSE-ESTANYOL M., L. RUIZ-AVILA, P. PUIGDOMENECH, 1992 A maizeembryo-specific gene encodes a proline-rich and hydropho-bic protein. Plant Cell 4: 413-423.

JÜRGENS G., 2001 Apical-basal pattern formation in Arabidopsisembryogenesis. EMBO J. 20: 3609-3616.

JÜRGENS G., 2003 Growing up green: cellular basis of plant de-velopment. Mech. Dev. 120: 1395-1406.

JÜRGENS G., R.A. TORRES RUIZ, T. BERLETH, 1994 Embryonic pat-tern formation in flowering plants. Annu. Rev. Genet. 28:351-371.

KAPLAN D.R., T.J. COOKE, 1997 Fundamental Concepts in the Em-bryogenesis of Dicotyledons: A Morphological Interpretationof Embryo Mutants. Plant Cell 9: 1903-1919.

KERSTETTER R., E. VOLLBRECHT, B. LOWE, B. VEIT, J. YAMAGUCHI, S.HAKE, 1994 Sequence analysis and expression patterns di-vide the maize knotted1-like homeobox genes into twoclasses. Plant Cell 6: 1877-1887.

KERSTETTER R.A., D. LAUDENCIA-CHINGCUANCO, L.G. SMITH, S. HAKE,1997 Loss-of-function mutations in the maize homeoboxgene, knotted1, are defective in shoot meristem mainte-nance. Development 124: 3045-3054.


KIESSELBACH T.A., 1949 The structure and reproduction of corn.Agricultural Experiment Station, University of Nebraska, Lin-coln, Nebraska 161.

KLINGE B., W. WERR, 1995 Transcription of the Zea mays home-obox (ZmHox) genes is activated early in embryogenesis andrestricted to meristems of the maize plant. Dev. Genet. 16:349-357.

KRANZ E., H. LÖRZ, 1993 In vitro fertilization with isolated singlegametes results in zygotic embryogenesis and fertile maizeplants. Plant Cell 5: 739-746.

KRIZ A.L., 1989 Characterization of embryo globulins encodedby the maize Glb genes. Biochem. Genet. 27: 239-251.

LEDUC N., E. MATTHYS-ROCHON, M. ROUGIER, L. MOGENSEN, P. HOLM,J.L. MAGNARD, C. DUMAS, 1996 Isolated maize zygotes mimicin vivo embryonic development and express microinjectedgenes when cultured in vitro. Dev. Biol. 177: 190-203.

LEE J.M., M.E. WILLIAMS, S.V. TINGEY, J.A. RAFALSKI, 2002 DNA ar-ray profiling of gene expression changes during maize em-bryo development. Funct. Integr. Genomics 2: 13-27.

LEE K., A.H. HUANG, 1994 Genes encoding oleosins in maizekernel of inbreds Mo17 and B73. Plant Mol. Biol. 26: 1981-1987.

LI Z.H., P.D. MATTHEWS, B. BURR, E.T. WURTZEL, 1996 Cloningand characterization of a maize cDNA encoding phytoenedesaturase, an enzyme of the carotenoid biosynthetic path-way. Plant Mol. Biol. 30: 269-279.

LID S.E., D. GRUIS, R. JUNG, J.A. LORENTZEN, E. ANANIEV, M. CHAM-BERLIN, X. NIU, R. MEELEY, S. NICHOLS, O.A. OLSEN, 2002 Thedefective kernel 1 (dek1) gene required for aleurone cell de-velopment in the endosperm of maize grains encodes amembrane protein of the calpain gene superfamily. Proc.Natl. Acad. Sci. USA 99: 5460-5465.

LONG J.A., E.I. MOAN, J.I. MEDFORD, M.K. BARTON, 1996 A mem-ber of the KNOTTED class of homeodomain proteins encod-ed by the STM gene of Arabidopsis. Nature 379: 66-69.

LUO M., P. BILODEAU, E.S. DENNIS, W.J. PEACOCK, A. CHAUDHURY,2000 Expression and parent-of-origin effects for FIS2, MEA,and FIE in the endosperm and embryo of developing Ara-bidopsis seeds. Proc. Natl. Acad. Sci. USA 97: 10637-10642.

MA Z., H.K. DOONER, 2004 A mutation in the nuclear-encodedplastid ribosomal protein S9 leads to early embryo lethalityin maize. Plant J. 37: 92-103.

MAGNARD J.L., T. HECKEL, A. MASSONNEAU, J.P. WISNIEWSKI, S. CORDE-LIER, H. LASSAGNE, P. PEREZ, C. DUMAS, P.M. ROGOWSKY, 2004Morphogenesis of maize embryos requires ZmPRPL35-1 en-coding a plastid ribosomal protein. Plant Physiol. 134: 649-663.

MANGELSDORF P.C., 1923 The inheritence of defective seeds inmaize. J. Hered. 14: 119-125.

MANSFIELD S.G., L.G. BRIARTY, 1991 Early embryogenesis in Ara-bidopsis thaliana. II. The developing embryo. Can. J. Bot.69: 461-476.

MANSFIELD S.G., L.G. BRIARTY, S. ERNI, 1991 Early embryogenesisin Arabidopsis thaliana. I. The mature embyro sac. Can. J.Bot. 69: 447-460.

MANSFIELD S.G., L.G. BRIARTY, 1992 Cotyledon cell developmentin Arabidopsis thaliana during reserve deposition. Can. J.Bot. 70: 151-164.

MATTHYS-ROCHON E., F. PIOLA, E. LE DEUNFF, R. MOL, C. DUMAS,1998 In vitro development of maize immature embryos: atool for embryogenesis analysis. J. Exp. Bot. 49: 839-845.

MAYER U., R.A.T. RUIZ, T. BERLETH, S. MISERA, G. JURGENS, 1991Mutations affecting body organization in the Arabidopsis em-bryo. Nature 353: 402-407.

MCCARTY D.R., T. HATTORI, C.B. CARSON, V. VASIL, M. LAZAR, I.K.VASIL, 1991 The Viviparous-1 developmental gene of maizeencodes a novel transcriptional activator. Cell 66: 895-905.

MECHIN V., T. BALLIAU, S. CHATEAU-JOUBERT, M. DAVANTURE, O. LAN-GELLA, L. NEGRONI, J.L. PRIOUL, C. THEVENOT, M. ZIVY, C. DAMER-VAL, 2004 A two-dimensional proteome map of maize en-dosperm. Phytochem. 65: 1609-1618.

MILLER E.C., 1919 Development of the pistillate spikelet and fer-tilization in Zea mays. J. Agr. Res. 18: 255-293.

MOL R., E. MATTHYS-ROCHON, C. DUMAS, 1993 In vitro culture offertilized embryo sacs of maize: Zygotes and two-celledproembryos can develop into plants. Planta 189: 213-217.

MOL R., E. MATTHYS-ROCHON, C. DUMAS, 1994 The kinetics of cy-tological events during double fertilisation in Zea mays L.Plant J. 5: 197-206.

MOLDENHAUER K.A.K., J.H. GIBBONS, 2003 Rice morphology anddevelopment. pp. 103-127. In: C.W. Smith (Ed.), Rice: Origin,History, Technology and Production. John Wiley & Sons, Inc.

NARDMANN J., J. JI, W. WERR, M.J. SCANLON, 2004 The maize du-plicate genes narrow sheath1 and narrow sheath2 encode aconserved homeobox gene function in a lateral domain ofshoot apical meristems. Development 131: 2827-2839.

NEILL S.J., R. HOGAN, A.D. PARRY, 1986 The carotenoid and ab-scisic acid content of viviparous kernels and seedlings of Zeamays L. Planta 169: 87-96.

NEUFFER M.G., W.F. SHERIDAN, 1980 Defective kernel mutants ofmaize: I. Genetic and lethality studies. Genetics 95: 929-944.

NIOGRET M.F., F.A. CULIANEZMACIA, A. GODAY, M.M. ALBA, M. PAGES,1996 Expression and cellular localization of rab28 mRNAand Rab28 protein during maize embryogenesis. Plant J. 9:549-557.

NORSTOG K., 1972 Early development of the barley embryo: finestructure. Am. J. Bot. 59: 123-132.

PETOLINO J.F., A.M. JONES, 1986 Anther culture of elite genotypesof maize. Crop Sci. 26: 1072-1074.

PILU R., G. CONSONNI, E. BUSTI, A.P. MACCABE, A. GIULINI, S.DOLFINI, G. GAVAZZI, 2002 Mutations in two independentgenes lead to suppression of the shoot apical meristem inmaize. Plant Physiol. 128: 502-511.

PLA M., J. VILARDELL, M.J. GUILTINAN, W.R. MARCOTTE, M.F. NIOGRET,R.S. QUATRANO, M. PAGÈS, 1993 The cis-regulatory elementCCACGTGG is involved in ABA and water-stress responsesof the maize gene rab 28. Plant Mol. Biol. 21: 259-266.

RANDOLPH L.F., 1936 Developmental morphology of the caryop-sis in maize. J. Agric. Res. 53: 882-916.


RIERA M., M. FIGUERAS, C. LOPEZ, A. GODAY, M. PAGES, 2004 Pro-tein kinase CK2 modulates developmental functions of theabscisic acid responsive protein Rab17 from maize. Proc.Natl. Acad. Sci. USA 101: 9879-9884.

RUIZ-AVILA L., S.R. BURGESS, V. STIEFEL, M.D. LUDEVID, P. PUIG-DOMENECH, 1992 Accumulation of cell wall hydroxyproline-rich glycoprotein mRNA is an early event in maize embryocell differentiation. Proc. Natl. Acad. Sci. USA 89: 2414-2418.

SANCHEZ-MARTINEZ D., P. PUIGDOMENECH, M. PAGES, 1986 Regula-tion of gene-expression in developing zea-mays embryos -protein-synthesis during embryogenesis and early germina-tion of maize. Plant Physiol. 82: 543-549.

SCANLON M.J., A.M. MYERS, 1998 Phenotypic analysis and molec-ular cloning of discolored-1 (dsc1), a maize gene requiredfor early kernel development. Plant Mol. Biol. 37: 483-493.

SCANLON M.J., P.S. STINARD, M.G. JAMES, A.M. MYERS, D.S. ROBERT-SON, 1994 Genetic analysis of 63 mutations affecting maizekernel development isolated from Mutator stocks. Genetics136: 281-294.

SCANLON M.J., A.M. MYERS, R.G. SCHNEEBERGER, M. FREELING, 1997The maize gene empty pericarp-2 is required for progressionbeyond early stages of embryogenesis. Plant J. 12: 901-909.

SCANLON M.J., D.C. HENDERSON, B. BERNSTEIN, R. JUNG, J.A.LORENTZEN, E. ANANIEV, M. CHAMBERLIN, X. NIU, R. MEELEY, S.NICHOLS, O.A. OLSEN, 2002 SEMAPHORE1 functions duringthe regulation of ancestrally duplicated knox genes and polarauxin transport in maize. Development 129: 2663-2673.

SCHEL J.H.N., H. KIEFT, A.A.M. LAMMEREN, 1984 Interactions be-tween embryo and endosperm during early developmentalstages of maize caryopses (Zea mays). Can. J. Bot. 62: 2842-2853.

SCHNEEBERGER R.G., P.W. BECRAFT, S. HAKE, M. FREELING, 1995 Ec-topic expression of the knox homeo box gene rough sheath1alters cell fate in the maize leaf. Genes Dev. 9: 2292-2304.

SCHOLTEN S., H. LORZ, E. KRANZ, 2002 Paternal mRNA and pro-tein synthesis coincides with male chromatin decondensationin maize zygotes. Plant J. 32: 221-231.

SCHULZ S.R., W.A. JENSEN, 1968a Capsella embryogenesis: theearly embryo. J. Ultrastruct. Res. 22: 376-392.

SCHULZ S.R., W.A. JENSEN, 1968b Capsella embryogenesis: theegg, zygote, and young embryo. Am. J. Bot. 55: 807-819.

SHERIDAN W.F., 1995 Genes and embryo morphogenesis in an-giosperms. Dev. Genet. 16: 291-297.

SHERIDAN W.F., M.G. NEUFFER, 1980 Defective kernel mutants ofmaize: II. Morphological and embryo culture studies. Genet-ics 95: 945-960.

SHERIDAN W.F., M.G. NEUFFER, 1982 Maize developmental mu-tants. Embryos unable to form leaf primordia. J. Hered. 73:318-329.

SHERIDAN W.F., Y.R. THORSTENSON, 1986 Developmental profilesof three embryo-lethal maize mutants lacking leaf primordia:ptd*-1130, cp*-1418 and bno*-747B. Dev. Genet. 7: 35-49.

SHERIDAN W.F., J.K. CLARK, 1993 Mutational analysis of morpho-genesis of the maize embryo. Plant J. 3: 347-358.

SMART M.G., T.P. O’BRIEN, 1983 The development of the wheatembryo in relation to the neighbouring tissues. Protoplasma114: 1-13.

SMITH L.G., D. JACKSON, S. HAKE, 1995 Expression of knotted1marks shoot meristem formation during maize embryogene-sis. Dev. Genet. 16: 344-348.

SOSSOUNTZOV L., L. RUIZ-AVILA, F. VIGNOLS, A. JOLLIOT, V. ARONDEL,F. TCHANG, M. GROSBOIS, F. GUERBETTE, E. MIGINIAC, M.DELSENY, P. PUIGDOMENECH, J.-C. KADER, 1991 Spatial andtemporal expression of a maize lipid transfer protein gene.Plant Cell 3: 923-933.

STIEFEL V., E.L. BECERRA, R. ROCA, M. BASTIDA, T. JAHRMANN, E.GRAZIANO, P. PUIGDOMENECH, 1999 TM20, a gene coding fora new class of transmembrane proteins expressed in themeristematic tissues of maize. J. Biol. Chem. 274: 27734-27739.

TIMMERMANS M.C., A. HUDSON, P.W. BECRAFT, T. NELSON, 1999ROUGH SHEATH2: a Myb protein that represses knox home-obox genes in maize lateral organ primordia. Science 284:151-153.

TRUE R.H., 1893 On the development of the caryopsis. Bot.Gaz. 18: 212-226.

VAN LAMMEREN A.A.M., 1986a A comparative ultrastructural studyof the megagametophytes in two strains of Zea mays L. be-fore and after fertilization. Agric. Univ. Wageningen Pap. 86:1-37.

VAN LAMMEREN A.A.M., 1986b Developmental morphology andcytology of the young maize embryo (Zea mays L.). ActaBot. Neerl. 35: 169-188.

VEIT B., S.P. BRIGGS, R.J. SCHMIDT, M.F. YANOFSKY, S. HAKE, 1998Regulation of leaf initiation by the terminal ear 1 gene ofmaize. Nature 393: 166-168.

VIELLE-CALZADA J.P., R. BASKAR, U. GROSSNIKLAUS, 2000 Delayedactivation of the paternal genome during seed development.Nature 404: 91-94.

VOGLER H., C. KUHLEMEIER, 2003 Simple hormones but complexsignalling. Curr. Opin. Plant Biol. 6: 51-56.

VOLLBRECHT E., B. VEIT, N. SINHA, S. HAKE, 1991 The develop-mental gene Knotted-1 is a member of a maize homeoboxgene family. Nature 350: 241-243.

WEATHERWAX P., 1919 Gametogenesis and fecundation in Zeamays as the basis of xenia and heredity in endosperm. Bull.Torrey Bot. Club 46: 73-90.

WEIJERS D., N. GELDNER, R. OFFRINGA, G. JURGENS, 2001 Seed de-velopment: Early paternal gene activity in Arabidopsis. Na-ture 414: 709-710.

WHITE C.N., W.M. PROEBSTING, P. HEDDEN, C.J. RIVIN, 2000 Gib-berellins and seed development in maize. I. Evidence thatgibberellin/abscisic acid balance governs germination versusmaturation pathways. Plant Physiol. 122: 1081-1088.


MAIZE EMBRYOGENESIS

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