endosperm development in zea mays; implication of gametic … · 2:1 (2m:1p) and it has been...

3089Development 121, 3089-3097 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Endosperm development in Zea mays; implication of gametic imprinting and

paternal excess in regulation of transfer layer development

W. L. Charlton1, C. L. Keen1, C. Merriman1, P. Lynch2, A. J. Greenland2 and H. G. Dickinson1

1Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK2Zeneca Seeds, Jealott’s Hill Research Station, Bracknell, Berks, RG12 6EY, UK

Fertilisation in maize (Zea mays), in common with mostangiosperms, involves two fusion events: one of the twosperm nuclei unites with the egg cell nucleus, while theother sperm nucleus fuses with the two central cell nucleigiving rise to the triploid endosperm. Since deviation fromthis nuclear ratio (2:1 maternal/paternal) in the endospermcan result in abortion, it has been suggested that thegenomes of the sperm and/or central cell are differentiallyimprinted during sexual development. By crossing anormal diploid maize line as female with its autotetraploidcounterpart, an unbalanced genomic ratio (2:2maternal/paternal) is created in the endosperm which oftenresults in the eventual abortion of the tissue. Detailed struc-tural comparison of these aberrant endosperms with

normal endosperms reveals that the formation of thetransfer cell layer, a tissue formed some 8 days after polli-nation and responsible for the transport of nutrients intothe endosperm, is almost completely suppressed under con-ditions of paternal genomic excess. The first structuralanalysis of the development of this tissue in normal andaberrant endosperms is reported, and the implications ofregulating the formation of such a tissue by gameticallyimprinted genes are discussed in the light of currenttheories on the consequences of genomic imbalance onearly embryonic development.

Key words: gametic imprinting, genomic imprinting, endosperm,transfer cells, Zea mays

SUMMARY

INTRODUCTION

In mammals it is now well established that the maternal andpaternal genomes transmitted to the zygote during fertilisationdiffer in gene expression during embryonic development, theexpression of a number of sequences being determined by theirparental origin. This parent-specific pattern of gene expressionis held to be established during gametogenesis by means of anepigenetic, reversible imprint (Reik et al., 1987; Sapienza etal., 1987). To be effective, this imprint must be capable ofsurviving mitosis, yet be removed during gametogenesis topermit reimprinting during passage through the germline of theopposite sex. How this gametic or genomic imprinting(Barlow, 1994) is achieved is not fully understood, but recentevidence from work on mice suggests that cytosine methyl-ation may play a major role in this process (Reik et al., 1987;Sapienza et al., 1987; Stöger et al., 1993; Feil et al., 1994; forreviews see Solter, 1988; Razin and Cedar, 1991; Riggs andPfeiffer, 1992; Surani, 1993), although other hypotheses havebeen proposed (Singh, 1994).

In plants, gametic imprinting has only convincingly beendemonstrated to influence development of the endosperm, atissue that nourishes the embryo (Kermicle, 1970; Lin, 1982,1984; Kermicle and Alleman, 1990; Chaudhuri and Messing,1994). Fertilisation in flowering plants involves the release oftwo sperm nuclei into the embryo sac; one unites with the eggto form the zygote, while the other fuses with the two haploid

nuclei of the central cell to form the triploid endosperm. Theendosperm thus possesses a maternal/paternal genomic ratio of2:1 (2m:1p) and it has been demonstrated (Lin, 1984) that thisratio can be crucial for the successful development of thistissue, any divergence resulting in abortion. Using an indeter-minate gametophyte (ig) mutant as the female parent, Lin(1984) was able to generate endosperms with ploidy levelsranging from diploid (2n) to nanoploid (9n) and, in all casesstudied, abnormal endosperms resulted unless amaternal:paternal genome ratio of 2:1 was maintained. Further,Lin (1984) was able to demonstrate unambiguously that theseeffects resulted from interactions between nuclear genomesand not from the influence of organellar genomes derived fromthe sperm and/or central cell.

The molecular and cellular consequences of these genomicinteractions are thus highly significant (for reviews seeBirchler, 1993; Matzke and Matzke, 1993). Most importantly,molecular mechanisms must exist by which both the copynumber and parental origin of certain genes are sensed and, incircumstances where the incorrect balance is detected, devel-opment of the early endosperm is aborted. The rapid degener-ation of these unbalanced endosperms makes analysis of themolecular events involved in gametic imprinting, and its con-sequences, very difficult. However, Lin (1984) reported a crossleading to an abortive endosperm which involved fertilising adiploid female plant with pollen from its autotetraploid toproduce tetraploid (2m:2p) endosperms. Interestingly, these

3090 W. L. Charlton and others

Fig. 1. Kernels of Zea mays following pollination by diploid andtetraploid plants. Top row: normal kernels resulting from diploid ×diploid pollination. Middle row: smaller kernels of near-normalappearance resulting from diploid × tetraploid pollination. Bottomrow: shrunken kernels resulting from diploid × tetraploid pollination.

endosperms appeared to undergo normal development until 10to 12 days after pollination (DAP), providing an opportunityfor the molecular and cellular analysis of the tissue prior to andduring abortion. The general cytology of aberrant endospermsof this type has been investigated (Cooper, 1951), but we reporthere a more comprehensive structural analysis of early devel-opment of normal and aberrant endosperms. In particular, wereveal the primary effect of paternal excess to be on the earlydifferentiation of the transfer tissue that links the base of thekernel with the embryo and endosperm. Molecular analysis ofthese events will form the subject of a later publication.

MATERIALS AND METHODS

Plant materialTwo lines of maize (Zea mays), a diploid cv. Wisconsin 23 (W23),kindly supplied by Dr Jane Langdale, and its autotetraploid cv.N107B, a gift from the Maize Seed Stock Centre (University ofIllinois, Urbana-Champaign, USA), were grown at Zeneca Seeds(Jealott’s Hill, UK) under glass with supplementary lighting andheating. Two crosses were performed: W23 × W23 and W23 ×N107B. The former provided control kernels while the latterresulted in a tetraploid endosperm formed by the fusion of the twohaploid central cell nuclei with a diploid sperm nucleus from thetetraploid N107B. Prior to silk emergence, ears were protected fromuncontrolled pollinations with shoot bags. As silks emerged, theywere trimmed back to the tip of the ear until sufficient silks wereexposed to ensure one application of pollen would produce a highlevel of fertilisation. Once pollinated, the ears were protected withtassel bags until harvested. All pollinations were performed in theearly morning.

Preparation of material for light and electron microscopyDissectionMaterial from both crosses was collected for study at 0, 2, 3, 4, 6, 8and 10 DAP. Following removal of the glumes from the kernel,embryo sacs or endosperm/embryo tissue at 0 and 2 DAP wereprepared for fixation by either repeatedly piercing the kernel pericarpwith a dissecting needle or removing a small section of the upper halfof the kernel epidermis. Endosperm/embryos from 3 and 4 DAP wereprepared by removing a half to two thirds of the nucellar tissue witha transverse cut being made close to the kernel base.Endosperm/embryo tissue at 6, 8 and 10 DAP was either completelyexcised from the nucellus or left seated in a cup of nucellar tissue tomaintain the integrity of structures linking the base of theendosperm/embryo assembly to the nucellus.

Fixation and embedding Tissue fixation was carried out for 4 hours at room temperature inKarnovsky’s fixative (4% paraformaldehyde, 3% glutaraldehydebuffered in 0.03 M phosphate, pH 7.0). After rinsing in buffer, thetissues were postfixed in 1% osmium tetroxide for 1-2 hours at roomtemperature before dehydration through a graded series ofacetone:water mixes (30%, 50%, 75%, 90%, 95%, 100%; each for 1hour) and embedding in medium grade Epon (TAAB) catalysed with1% accelerator (TAAB). Prior to embedding in Epon, the tissue wassupported in 1% low melting point agarose (Flowgen Instruments Ltd)buffered with 0.03 M phosphate, for protection against mechanicaldamage during resin impregnation.

Sectioning Semithin sections (7 µm) were cut on a 5000 MT Sorvall ultramicro-tome, stained with 1% Toluidine blue in 1% borax and/or Periodicacid/Schiff’s reagent (PAS) (Feder and O’Brien, 1968) and pho-

tographed with a Zeiss Axiophot light microscope on Pan F film.Following light photography, the semithin sections were re-embeddedfor ultrathin sectioning at 90 nm. Ultrathin sections were stained withuranyl acetate and lead citrate and examined using a JEOL 2000EXtransmission electron microscope operating at 80 kV.

RESULTS

Pollinations with pollen from tetraploid parentsFertilisation with pollen from tetraploid parents proved poorwhen compared with pollen from diploid parents. While thelatter regularly produced fully fertilised ears, it was notuncommon to find ears of the former completely devoid ofdeveloping kernels or containing as few as 5-10 kernels. Onlyoccasionally would more than 50% of ovules be successfullyfertilised.

Pollinations that were successful gave rise to two pheno-types: abundant but shrivelled kernels, or a low number ofsmall but well-formed, ‘normal’ kernels (Fig. 1). Although aninsufficient number of ears were allowed to develop tomaturity to permit a statistical analysis of these phenotypes,Randolf’s earlier work (1935) reported that less than 0.5% ofthe successful 2n × 4n pollinations gave rise to ‘normal’kernels. The earlier work by Randolf also reported that approx-imately 10% of the well-formed ‘normal’ kernels and very fewof the aborted kernels produced viable seedlings.

Development of triploid and tetraploid endosperms0-6 DAP‘Fertilisation’ of the binucleate central cell by a haploid spermnucleus initiates endosperm development following a pathwayfirst described by Cooper (1951) (see also Fig. 2). By c.6 DAP,the tissue has become fully cellular and, through repeateddivisions, has increased in volume approximately 3 fold (Kies-selbach, 1980) to assume a characteristic cone-shaped

3091Gametic imprinting and endosperm development

Fig. 2. Summary of the development of Zea mays endospermsformed following pollination of a diploid seed parent by a diploidpollen source (2n × 2n), and by a tetraploid source (2n × 4n).Structural development of the two types of endosperm appearsidentical until c.5 DAP.

3A

structure. Under the light microscope little difference can beobserved at this stage between the triploid and tetraploidendosperms, apart from a tendency for the upper third of thetetraploid endosperm to narrow more sharply.

During the development of the outermost basal cells of thetriploid endosperms, fine wall ingrowths appear (Fig. 3A,B),signalling the first stages in transfer cell development (Kies-selbach and Walker, 1952; Kiesselbach, 1980). The devel-oping ingrowths, which are principally restricted to the outerand tangential cellular faces, are frequently observed in asso-ciation with cytoplasmic inclusions containing an electron-lucent matrix similar in appearance to that in the developingwall (Fig. 3A,B). The cytoplasm of these cells appears veryactive, being particularly rich in small, spherical mitochon-dria. Importantly, little or no development of the ingrowthstakes place in the outermost basal cells of the tetraploidendosperms. In circumstances where slight developmentdoes take place, apparently random and malformed aggre-gations of wall material are laid down (Fig. 4). Here, the wallmatrix itself is different, being more electron-opaque (Fig.4). The cytoplasm surrounding these early wall inclusions isdisorganised with few mitochondria. Additionally, whiletriploid endosperms appear firmly anchored at their bases tothe nucellar tissue, tetraploid endosperms are readilydetached.

Development of triploid and tetraploid endosperms6-10 DAPBy 8 DAP the outermost basal cells of the triploid endospermshave become elongated, and their wall ingrowths readily observ-able under the light microscope (Fig. 5). The invaginationsappear tubular in section and are about 0.5-1.0 µm in diameter;there are c. 1-3 per 10 µm2 of the cell surface and they extendsome 5-10 µm into the cytoplasm (Figs 6, 7). They continue tobe associated with inclusions containing a wall-like matrix and,again, the surrounding cytoplasm contains numerous mitochon-dria. By contrast, corresponding cells in the tetraploidendosperms remain devoid of maturing wall ingrowths. Thecytoplasm of these cells appears inactive and frequently becomeshighly vacuolate; in later stages cytoplasmic bridges may beobserved (Fig. 8A). In addition, the narrowing of the apical region

Fig. 3. (A,B) Electronmicrographs of grazingsections of the outer surfaceof the transfer layer, 6 DAPof a diploid plant by diploidpollen. Ingrowths (I) arebeginning to develop,frequently associated withcytoplasmic vesicles (V).The cells are interconnectedby plasmodesmata (arrows).Profiles suggestive ofvesicular contribution to theingrowths are shown in A.Scale bars, (A) 500 nm; (B) 1 µm.

B


4

Fig. 4. Electron micrograph of outer face of transfer cells in atetraploid endosperm, 6 DAP. No organised ingrowths have formed.Instead the cytoplasm appears to be discharging irregular masses (M)into the ‘nucellar’ surface of the wall (W). Scale bar, 500 nm.

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Fig. 6. Electron micrograph of ingrowths, more highly developedthan those shown in Fig. 3, in endosperm 8 DAP by a diploid plant.Note the high density of mitochondria (arrows) in the cytoplasminvesting these structures. Scale bar,1 µm.

of the tetraploid endosperms has, by 8 DAP, become more pro-nounced when compared with triploid endosperms. Importantly,the inner cells of the tetraploid endosperms begin to degenerateat this stage. Originating in a mass of cells at the centre of theyoung endosperm, changes normally associated with necrosistake place; these include disintegration of the tonoplast, intensevesiculation, the accumulation of electron-opaque inclusions and,eventually, the condensation of nuclear material. Despite thisapparent cellular degeneration, no clear difference may be

Fig. 5. Light micrograph of developing ingrowths of the outer wallsof transfer tissue cells, 8 DAP by a diploid pollen source. PASstaining reveals the ingrowths to contain high levels of carbohydrate.Scale bar, 15 µm.

detected either in endosperm length (approximately 2.7 mm) orwidth (approximately 1.5 mm) between triploid and tetraploidendosperms at 8 DAP.

Differentiation of the aleurone layer in the triploid tissues isobserved between 6 and 10 DAP, with the accumulation ofspherosomes and protein bodies in the outer layers of theendosperm (Fig. 9). While these cell layers contain normalcytoplasm in tetraploid endosperms, protein bodies and spher-osomes are absent (Fig. 10).

Wall ingrowths of triploid endosperms cover a large pro-portion of the placentochalazal region by 10 DAP (Fig. 7), andextend to the junctions between cells of the endosperm up tothree cell layers beneath the superficial cell layer. The matrixcomposing these wall ingrowths is continuous with the wallsof the nucellar cells and thus presumably contributes to thehigher mechanical strength of this region when compared withkernels containing tetraploid endosperms. No ingrowths arepresent at the nucellar face of tetraploid endosperms (Fig. 8B).

In tetraploid endosperms 10 DAP, the boundary of theregion of cellular dissolution approaches the periphery of thetissue, with no further development of the transfer cell layer.By contrast, triploid endosperms at 10 DAP are plump, fullycellular tissues, and at this stage the first evidence of a size dif-ference between the two types of endosperm can be distin-guished. On average, 10 DAP triploid endosperms are twice aslong and twice as wide as tetraploid endosperms. Further,tetraploid endosperms assume a deflated appearance with theirsurfaces becoming increasingly infolded.

Starch development in triploid and tetraploidendosperms 0-10 DAPApart from differences in the development of the aleuronelayer described above, surprising differences also occur in theaccumulation of starch in the interior of the endosperms. By


Fig. 7. Electron micrograph of glancing section of the outer surface of the transfer tissue, as in Fig. 6, but from material 10 DAP. Extensiveingrowths are present in all cells, and in some regions the cytoplasm between the ingrowths (arrows) is highly reduced. Scale bar, 1 µm.

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contrast with events at the tissue surface, PAS staining revealsthat tetraploid endosperms accumulate starch at an earlier stagethan triploid tissues, small starch grains becoming visible intetraploid cells as early as 8 DAP, whereas the first appearanceof starch occurs in triploid tissues at 10 DAP (data not shown).

DISCUSSION

By crossing a female diploid with a male autotetraploid, wehave generated tetraploid endosperms with a genomic ratio of2:2 and like Lin (1984) found abortion to occur during devel-opment. However, by contrast with reports of completeabortion of tetraploid endosperms by maturity (Lin, 1984), asmall number of the endosperms produced from our 2n × 4ncrosses were normal, plump tissues, albeit slightly smaller thanthe wild type. Similar observations have been previouslyreported by Randolf (1935) and Cooper (1951), but not in thecontext of genomic balance. Such ‘normal’ kernels are unlikelyto be the result of contamination by pollen from a diploid plantor a reversion of tetraploid to diploid pollen as the triploidstatus of plants derived from these apparently normal kernelsstudied by Randolf (1935) was confirmed cytologically. It isthus important to determine whether the dramatic develop-mental changes reported relate to the near-normal or to the verysmall, shrunken kernels. However, over the course of thisstudy, a sufficiently large number of kernels from each devel-opmental stage was examined to indicate strongly that theabsence of transfer tissue is associated with the developmentof the very small, shrunken kernels. We believe the largerkernels do possess transfer cells, although this has not beenchecked during our studies.

Following fusion of the two central cell nuclei with a sperm

nucleus approximately 24 hours post pollination, both triploidand tetraploid endosperms commence parallel developmentalpathways until approximately 6 DAP when, in the triploidendosperm, early signs of transfer cell development are firstobserved. In the tetraploid endosperms, this development failsto be fully initiated. The wall ingrowths, which first identifythe transfer cells, are contiguous with the walls of cells formingthe placentochalazal region and, early in their differentiation,appear to assume an anchorage role, holding the developingendosperm to the base of the nucellus. Although the first signsof transfer cell differentiation may sometimes occur in thetetraploid endosperms, development clearly does not progressto the stage where anchorage occurs; for this reason theseendosperms are easily detachable by microdissection. By 8DAP in triploid endosperms, the wall ingrowths of the transfercells are readily observed under the light microscope and by10 DAP the majority of the endosperm cells adjacent to theplacentochalazal region possess these ingrowths, as do cells upto three layers beneath the principal transfer cell layer.

Since Harz first reported this presumably nutrient-transport-ing tissue for maize in 1885 (cited by Kiesselbach, 1980), thislayer has been described in some detail by Harrington andCrocker (1923) in Johnson grass (Holcus halepensis L.), inSaccharum by Artschwager and co-workers (1929), in Coix byWeatherwax (1930), and in maize by Kiesselbach and Walker(1952), Kiesselbach (1980) and Felker and Shannon (1980),who first recognised the presence of transfer cells in this tissue,and by Schief and co-workers (1984). Transfer cells occur inall major taxa, and although normally associated with thetransfer of nutrients from sporophyte to gametophyte, arefound in many other situations (Gunning and Pate, 1969),occurring in a diversity of tissues and being involved in thetransfer of a wide range of solutes. Even within the reproduc-


8A

B

Fig. 8. (A) Electron micrograph of outer face of transfercells in a tetraploid endosperm, 10 DAP. No organisedingrowths have formed and there is little development ofthe outer (nucellar) wall (W). Cells appear to be joined vialarge cytoplasmic bridges (arrows). Scale bar, 5 µm. (B) AsA but at a higher magnification. The outer (nucellar) wallappears to contain compacted masses (arrows) of electron-opaque material. Scale bar, 1 µm.

tive structures of the Gramineae, this tissue is not restricted tothe endosperm, for studies of the wheat caryopsis (Smart andO’Brien, 1983) revealed the development, at 15 DAP, of alayer of cells with wall ingrowths within the nucellar epidermalcells at the base of the kernel. This interesting observationsuggests that transfer cells in this region are capable of per-forming both export and import functions.

Interestingly, Lyznik and co-workers (1982) have shownthat translocation of amino acids into the endosperm, presum-ably via the transfer cells, takes place against a concentrationgradient, created predominantly by high levels of alaninepresent in the tissue. The energy requirements for the transportof amino acids and other nutrients, combined with the absenceof an apoplastic pathway between the sporophytic nucellus andgametophytic endosperm via plasmodesmata, may wellexplain the very large numbers of mitochondria in the transfertissue. Other physiological studies (Shannon, 1972; Shannon

and Dougherty, 1972; Felker and Shannon, 1980) have shownthat during nutrient uptake by the endosperm, sucrose isunloaded from the pedicel parenchyma into the apoplast of theplacenta-chalazal tissue. It is then either hydrolysed by acidinvertase(s) into fructose and glucose, prior to uptake by thetransfer cell layer into the endosperm symplast (Felker andShannon, 1980; Doehlert and Felker, 1987), or transferred tothe basal cells of the endosperm as unhydrolysed sucrose(Porter et al., 1985, 1987) to be hydrolysed by invertases in theendosperm.

The apparent lack of a nutrient transport tissue in tetraploidendosperms is consistent with the observations by Cooper(1951) of ‘undifferentiated basal cells’ in tetraploid and pen-taploid endosperm resulting from 2n × 4n and 4n × 2n crosses.Although Cooper (1951) states that the ‘basal layer of cellsfails to differentiate in a normal manner’, it is never explicitlystated that an absorbing tissue fails to form. Further, in his


Fig. 9. Electron micrograph ofsurface cells of a normalendosperm, 10 DAP, in theregion subsequently occupiedby the aleurone layer. Largenumbers of spherosomes (S)are present. Scale bar, 2 µm.Fig. 10. As Fig. 9, butmaterial from a tetraploidendosperm. No spherosomesare visible. Scale bar, 1 µm.

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summary, failure of the basal layer is described only for thetetraploid endosperms, with no mention of the pentaploidendosperms from the 4n × 2n crosses. Since, by 16 DAP, thepentaploid endosperms are packed with starch (growth ceasingonly at later stages) aberrant development is unlikely to resultfrom the lack of transfer tissue.

After uptake by the transfer layer cells, nutrients are translo-cated into the rapidly expanding endosperm by a core ofelongated cells extending from the transfer layer through thecentral region of the endosperm and into the apex (Weather-wax, 1930; Lampe, 1931; Brink and Cooper, 1947; Kiessel-bach, 1980). No evidence of this conducting tissue is seen inthe tetraploid endosperms at 10 DAP. Rather than containingspecialised conducting tissue, this region is occupied by appar-ently necrotic cellular debris.

Our observations suggest that the departure from the normal2m:1p genomic ratio in the maize endosperm results in thealmost complete suppression of the development of the transfercell layer. Other abnormalities found, such as cellular necrosisand retarded aleurone development, may merely be a conse-quence of nutrient starvation resulting from the absence of thetransfer tissue. The precocious appearance of starch in thetetraploid endosperms is, however, less easily explained.

It is possible that suppression of transfer cell developmentresults indirectly from changes in maternal tissue, which couldrestrict the uptake of nutrients essential for this complexcellular differentiation. Such an effect has been reported byMiller and Chourey (1992). Using the miniature 1 mutant firstdescribed by Lowe and Nelson (1946), they showed that, inits homozygous condition (mnmn), the mutation produces aninvertase deficiency in the endosperm resulting in the degen-eration of the placentochalazal cells and their eventual sep-aration from the endosperm basal cells. The degeneration ofthese cells in turn causes a loss of invertase in this region. Acombination of the structural separation, and a lack ofinvertase in both the basal cells and the placento-chalazalregion, effectively prevents the uptake and processing ofnutrients by the endosperm, causing a shrunken phenotype.There is, however, very little evidence that similar eventsoccur in the tetraploid endosperms for, while we did not study

the maternal tissue of the tetraploid-containing kernels beyond6 DAP, neither Cooper’s (1951) study of tetraploidendosperms, nor Brink and Cooper’s (1947) report of the de17mutant – which produces a phenotype similar to that of thetetraploid endosperms – described any disruption of maternaltissue.

Animal studies reveal that androgenetic mouse embryos(0m:2p) develop poorly, yet have well developed extra-embryonic membranes (Barton et al., 1984); paternal excess inhumans (1m:2p) leads similarly to under-developed foetuseswith large placentas (Hall, 1990). Gynogenetic and partheno-genetic mouse embryos (2m:0p), and maternal excess inhuman foetuses, result in a reverse situation - with smallerplacentas than normal. These studies have led Haig andWestoby (1989) to propose an elegant theory of parental ‘tug-of-war’ to explain the relationship between gametic imprintingand embryo development. The theory holds that selectionfavours the imprinting of certain genes from a male so that theyensure maximum investment from the female towards hisoffspring during development, while the female genes areimprinted to limit her investment in particular offspring.Although the transfer tissue of maize may reasonably be con-sidered analogous to the placenta of mammals, our observa-tions suggest that this theory fails to hold for endospermimprinting in plants for, if Cooper’s (1951) results are taken atface value, both maternal and paternal excess give rise to adeficient ‘placenta’. This would indicate that it is not paternalexcess per se which causes this deficiency but rather anydeviation from the 2m:1p ratio. On the other hand, if penta-ploid endosperms (Cooper, 1951) do produce a transfer layer,as some evidence suggests, the situation conflicts directly withHaig and Westoby’s (1989) theory - for seemingly seed devel-opment is favoured despite maternal excess.

Whether paternal excess alone or genomic imbalance resultsin the phenotype observed, at least one of the genes involvedin transfer cell development must be differentially imprinted.Since genomic imbalance can suppress the development of thecomplete transfer tissue, the imprinted genes are likely to beregulatory in function and act at an early developmental stage.On the basis of these assumptions, a simple model (see Fig. 11)


Fig. 11. A simple model to explain the regulation of endospermdevelopment through gametic imprinting. In the normal triploidendosperm, genes A and B are transcribed from the paternallydonated genome to produce gene products A and B. The expressionof A is suppressed in the maternally donated genomes throughgametic imprinting, and thus only the maternal B gene product issynthesized. It is suggested that only a combination of A and Bpolypeptides, in the correct ratio, can activate development.

for their operation can be proposed on the basis of the centralcell nuclei but not the sperm nuclei carrying imprinted loci, withimprinting suppressing gene expression in the endosperm.Thus, a regulatory protein (A), transcribed from the paternalallele (A) and not from the imprinted maternal allele, complexeswith a second regulatory protein (B) transcribed from both thematernal and paternal loci (B). Depending upon the activities ofA and B, a ratio of A and B would be generated which resultsin an active complex. Upsetting the genomic balance woulddisturb the ratio of A and B and thus affect the activity of theAB complex resulting, eventually, in the arrest of development.Such concentration-dependent activation and repression hasrecently been shown to exist in Drosophila by Sauer and Jäckle(1993) for, at a certain concentration, the zinc finger-type tran-scription factor Kruppel (Kr) functions as a transcriptionalactivator in its uncomplexed form; however, at higher concen-trations, homodimerisation of the Kr protein transforms it intoa transcriptional repressor. Interestingly, the de17 mutation firstdescribed by Brink and Cooper (1947) may involve just such aregulatory gene responsible for the initiation of transfer tissuedevelopment. The phenotype of this mutant includes shrunkenkernels and lack of development of the basal endosperm cells.Further, the description of the undifferentiated basal cells of thismutant is remarkably similar to our observations for the corre-sponding cells of the tetraploid endosperm. While this modelwas proposed to explain specific observations in maizeendosperms, it is also applicable to other circumstances where‘imbalance’ in the number of endosperm nuclei affects devel-opment, such as in the Solanaceae (Hawkes and Jackson, 1992).

There are important reasons why mechanisms have evolvedto preserve the genomic ratio in the endosperm. Clearly thesystem will prevent the spontaneous development of theendosperm in the absence of pollination, and equally anydevelopment resulting from the fusion of both sperm nucleiwith the two polar nuclei. More importantly it must also restrictthe success of agamospermous embryos - thus protecting

sexual reproduction (Haig and Westoby, 1991). Interestingly,in outcrossing plants this mechanism would also act tosuppress polyploidy, which is held to be a major source ofgenetic variation in the process of evolution (Stebbins 1950).However, these aims would also be achieved were the embryoitself to be imprinted, and the reason for the imprinting of theendosperm rather than the embryo probably lies in the repro-ductive strategies adopted by the angiosperms: while it isimportant to maintain a strong selection for sexual reproduc-tion, it is advantageous for plants to retain the option of asexualreproduction should the need arise. By imprinting theendosperm, selection for sexual reproduction is maintained,and by keeping the embryo free from imprinting, the optionstill exists for asexual reproduction. Alternatively, it maysimply be that the process of imprinting carries a cost in evo-lutionary terms; thus by imprinting the endosperm only, thiscost is lessened yet sex still ensured.

This work was funded under the UK BBSRC Stem Cell Initiative.The authors wish to thank Kelly Pritchard, Stephen Moore and JohnBaker for technical assistance and Ann Rogers and Melissa Spielmanfor help in preparing the manuscript.

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371-380.

(Accepted 16 May 1995)

endosperm development in zea mays; implication of gametic … · 2:1 (2m:1p) and it has been...

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