Development composition of spinach ovule of angiosperms consists enveloped by integuments ...

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Acta801.Neerl. 29(4),August1980,p.243-260.
Development and composition
of the spinach ovule
H.J.Wilms
VakgroepPlantencytologieen-morfologie,Botanisch Laboratorium,Landbouwhogeschool,Arbo-
retumlaan 4,6703 BD Wageningen
SUMMARY
The ultrastructure and histochemistryofthe developingspinach ovule have beenexamined. The
developmentanddifferentiation oftheinteguments,nucellus and femalegametophyteresultsinan
ortho-amphitropousorganisationofthe ovule.Inthenucellus four partscanbe distinguished:the
conductive part,theoriginalchalazalpart,thechalazal proliferatingpart and thelaterial part.The
cellsofthevariouspartshavecommonfeaturesaswellasdistinguishingcharacteristicsrelated totheir
positionandfunction.Theinner andouterintegumentsshowdifferent featuresduringtheirdevelop-
ment.Intheouterintegument3-5celllayersdevelop,buttheinnerintegumentshowsadevelopment
oftwo differingcelllayers.Contactbyplasmodesmatabetweenthe two cell layersdiminishes and
stops atmaturity. The localization ofdifferentreservesubstances (starch, otherpolysaccharides,
proteins,lipids)have beenstudiedinthedevelopingovulestodeterminethe nutritionalsupplyofthe
embryosacand embryo.Extensivechangesintheamountofreservesubstanceshavebeen observed
in the ovule right up tomaturity ofthe embryosac. In the outerintegumentstorage ofstarch
increases much uptomaturityand afterfertilization a gradualdecrease occurs. Thestorage and
transfer function ofsomecell types in relation tothe nutrition oftheembryosacand embryois
discussed.Thepresence andlocation ofpolysaccharidesduringmegasporogenesisandmegagameto-
genesisisconsidered.Finally,thepossiblepathwayofmetabolitesin spinachduringdifferentphases
ofthedevelopingovule isdiscussed.
1. INTRODUCTION
The ovule ofangiosperms consists of the nucellus enveloped by one or two
integuments, and is attached to the placenta by a short stalk, the funiculus.
Ovules can be classifieddepending upon the degree ofcurvature during ovule
development (Schnarf 1929 and Maheshwari 1950, 1963). Apart from this
morphological variation, ovulesalso can highly vary ininternalstructure.The
variationinsize, dimensionsand histology ofthenucellusis muchgreaterthan
thedistinctionbetweentenui-andcrassinucellatetypesuggests (Bouman 1974).
Thereis astrongvariationinnumberofcellsinthevarioustissuesandindegree
ofdifferentiationofthe variouscellsandtissues.Aspecific problemwithrespect
to the formationand development ofthe ovule is its nourishment.Nutrition
mainly takes place from the placenta through the funicular vascular tissue.
Transport occurs in the ovule through micellar cells to the developing female
gametophyte or embryo andendosperm. According to Jensen(1963, 1965a) in
cottonthe synergids playarole inthenutritionofthefemalegametophyte. Van
Went(1970) forPetunia Mogensen(1972) forQuercus andNewcomb(1973a,
,
b) forHelianthussuggestdifferentroutes ofmetabolitesto the embryo sac and
developingembryo. However,inthisdiscussiononly thenutritionoftheembryo
244 H. J.WILMS
sac hasbeenconsidered. No considerationhasbeengiven to the otherparts of
the ovule.
Thepresentreportdealswiththe study ofthedevelopment and organisation
ofthe cellsoftheSpinacia oleraceaL. ovule, attemptingto elucidatethewayof
bending andthepathwayofsomemetabolites.Itispartofaninvestigationofthe
early embryogenesis in spinach in terms of cellular ultrastructure and
composition.
2. MATERIALS AND METHODS
Plants of Spinacia oleracea L., cv. Prevital, were grown in the greenhouse at
approximately 25 C. Dissectedovuleswere fixedaccording to oneofthefollow-
ing procedures.
A.Fixationfor 1.5hrsin2.5%KMn0 in0.05 Mphosphate bufferatpH 7.2 at
4
room temperature.Afterfixationthetissuewaswashedin buffer, dehydrated in
a graded ethanolseries andembeddedin ERL.
B.Fixationin3.5% glutaraldehyde in 0.2M phosphate bufferat pH 7.2for3.5
hrs at room temperature. After fixationthe tissue was washed several times
during5 hrs inbufferand subsequently post-fixed overnight in2% 0s0 in 0.2
4
Mphosphate bufferatpH 7.2atroom temperature.Thefixedmaterialwasthen
washedinbuffer, dehydrated in agraded'ethanol seriesandembeddedinEpon.
Post-staining with 2% uranylacetate occurred in the 70% ethanol for 1 hr.
Sectionswere post-stained with0.5%leadcitratefor 1minute(Reynolds 1963).
Tissueusedforhistochemical localizationofstarch wasfreeze-fixedinaglycol
medium, cut in a IEC CTFcryo-microtome at —10 C and stained with IKI
(Jensen 1962).Tissueforotherhistochemicallocalizationprocedures was FAA-
fixed and sectionedin paraffin wax. Forinsoluble carbohydrates the periodic
acid-Schiff(PAS) and for proteins thechloramineT-Schiffstaining was used
(Jensen 1962).
3. RESULTS
The large megaspore mother cell of spinach is surrounded by multilayered
nucellustissue (figs. I, 2). Round themicellar tissue theinteguments develop.
Early in ovular development the nucellus points upward, but as it grows it
gradually bendsdownandfinally becomesortho-amphitropous {fig. 4A).
3.1. Integuments
Formationofthe integuments starts beforethe megasporemothercell reaches
theleptotene stage. Theoriginofbothinteguments is dermic.Theinnerintegu-
ment initiatesfirst, followedshortly by theouter.During itsontogenytheinner
integument is twocells thick, except atthe base and atthe micropyle where it
becomesthicker (fig. 3). Ina youngstageofdevelopment thecellsoftheouter
layer are square and those of the inner layer are rectangular in longitudinal
sectionsoftheovule(fig.5). Plastidswithfewthylakoids areregular,intheouter
DEVELOPMENTOFTHE SPINACHOVULE 245
Figs. 1-3.EarlystagesinovuledevelopmentinSpinacia.Thethick lines indicatetheborderbetween
dermallyandsubdermallyoriginatingcells.Double endedarrowsindicate growthpatterninthenu-
cellus. Thedirection ofthearrowsindicates thedividingdirection. Thelengthofthearrowsindicates
divisionactivity.Fig. 1.Cellpatternintheintegumentsandnucellus atmegasporemothercellstage.
Fig, 2.Ovuleatmegasporemothercellstagejustbesidethemegasporemothercell.Differentdivision
activitiescanbe seenin thenucellus. Fig. 3.Ovule with theembryosacin theeightnucleate stage.
246 H. J.WILMS
layer sometimeswithstarch. Dictyosomes with 5-7 cisternaeare relatively fre-
quent.The cisternae have an average length of0.6 pm and form many small
vesicles. Thecellwallbetweenthetwolayers oftheinnerintegument isconstant
in thicknessexcept at the places with plasmodesmata. The walls between the
individualcellsofeachlayer differinthickness andallhaveplasmodesmata. Up
to maturityofthe ovulethecellsofbothlayers elongate,whileinthe innerlayer
stillsome divisionsoccur. Thenumberofplasmodesmata betweentheindividual
cells ofeachlayer remains the same. Cells ofthe outer layer reach a length of
about30pm,whichistwicethelengthofthecellsoftheinnerlayer. Thethickness
of the total innerintegument remains about8 pm. The surface of the inner
integument iscovered withacuticle.Wherethiscuticlebordersthenucellus, itis
less compact.
Theouterintegument is initiallyslightly shorterthantheinnerone(fig. 1). As
theovule growsthe outerintegument becomesequal in length bymorefrequent
celldivision(fig. 3). Bythistimetheouterintegument is composed of2-5layers
ofslightly radially elongated cells (fig. 3).Thecells possess somelipid granules
and many plastids (fig. 6). Initially no starch is observed in the plastids, but
towardsmaturitytheamount and sizeofstarch grains increasesinthe plastids
(fig. 7). Cellsoftheouter layer containalarger amount ofstarch. Intercellular
spacesappearbetweenthesubsequent layers (figs. 4G, 7).Theircontent showsa
slight homogeneous electron-density. After maturityand untilfertilizationthe
amount ofstarch increases strongly in the outerintegument.
After fertilizationa decrease ofstarch inthe outer integument occurs. The
wallsbetweenthe outer integument cellsarerelatively thin withmany plasmo-
desmata, especially in the radialwalls. The surface ofthe outer integument is
covered witha distinctcuticle.
3.2. Nucellus
At the leptotene stage ofthemegasporemothercell thegrowth ofthe nucellus
becomes unequal. Unequal growth isachieved by a variationin bothcell elon-
gationandcelldivision activity (fig.I). Thecellscloseto theinnerbending side
are lesselongated thantheothercells.Thelatteralso dividemorefrequently(fig.
I).Attheouterbending sidesomepericlinal divisions occur (fig. I). Initially the
expansion oftheovuleis merelyduetocelldivision.Obliquely orientatedfilesof
cellsare produced (fig. 2).At this stageallnucellus cells showthe same ultra-
structure. At the megasporestage thefirst differences betweenmicropylar and
chalazalcellsareobserved(fig. 8).Themicropylarcellshaveamyloplasts, many
dictyosomes andvacuoles,andthechalazalcellshavemanymitochondria.Inthe
micropylar tissuetheoriginally thinwallsthicken(fig. 8).Thegeneral processes
of cell elongation and divisioncontinue during thenext developmental stages
(jig. 3). Theultimateovuleshape is highly definedby the formationoffiles of
cellsatthechalazalsideofthemegasporeor youngembryo sac. Thesefilesbend
fromthe funiculusto themegaspore.Thistogether withthedivisionand exten-
sion ofthe cellsat the micropylar side ofthe megasporeresults ina long and
curvedovule.
DEVELOPMENTOFTHESPINACHOVULE 247
Fig. 4.A. Cellpattern in thematureovule. Thedottedlines mark thevariouspartsofthenucellus.
B-G:Cytologicaldrawingsofcelltypesofthenucellusandinteguments.B. Originalchalazal cell.C.
Transmittingcelloftheconductivetissue. D. micropylarcell oftheconductive tissue.E.Chalazal
proliferatingcell. F.Lateralcell and G.Innerand outerintegument.
248 H. J.WILMS
During laterstages inovule development variousparts ofthenucellusshow
specific differentiations. Most of the elongation of the ovule is due to cell
enlargement, whichis especially prevalentinthe micropylar part, inthecentral
corearoundtheembryo sac andinthe originalchalazalnucelluscellsbehindthe
embryo sac (fig. 4A).Cell divisionoccurs in thenucelluscellsperpendicular to
theplacenta andintheextensionofthefuniculus,whichformmanyradialfilesof
cells.Theseactivitiesgive themature ovuleitsfinalshape(Jig. 4A). According to
theplace, structureandorganisation ofthevariousparts ofthemature nucellus
thefollowing tissuescan be distinguished: a.the original chalazaltissuebehind
the antipodal side of the embryo sac, b. the conductive tissue between the
micropyleandtheembryosac,c. thechalazalproliferatingtissueintheextension
ofthe funiculusand d. the lateraltissue. Therelative position ofthesevarious
tissues is indicatedinfig. 4A.
Theoriginalchalazaltissue.Thegeneralformoftheindividualcellscanbe seen
infig.4B.Thecellwallsarerelativelythinandespecially theshortendwallsshow
ahigh plasmodesmata density(fig. 10).Thelength ofthecellsintheyoungstage
is 3-5 pm, whichis one quarterofthe width (12-17 pm). In the mature stage
however,thelength isasmuchas 125pm,whichisabout20timesthewidth.The
nucleusremains locatedat the centre ofthe cell accompanied at bothsides by
vacuoles. Oval mitochondriawith manywell developed cristae are abundant
(fig. 10).Ovalto pear-shaped plastidsarepresentwithsomesingle thylakoids. In
theyoungstagemanysmallstarchaccumulationsarepresent(fig. 9), whereasat
maturity there are hardly any (fig. 17). Endoplasmic reticulum(ER) is first
observedasshortcisterns, butlatergroupingoflongcisterns withsomebranch-
ing is observed in the peripheral cytoplasm. The amount of dictyosomes de-
creases fromabundantintheyoungstage torare at maturity.
Theconductivetissue. Itconsists oftwo celltypes (fig. 4A). At the micropylar
endare themicropylarcellswithonly slightlythickened walls(fig. 4D).Nearthe
embryo sac are the small, so-calledtransmitting cellswith thick cell walls(fig.
4C). The structural differentiationof the cells of theconductive tissue starts
shortly before the ovule development has reached the functional megaspore
stage (fig. 8). Then their walls thicken and the plastids accumulatestarch for
sometime.
At maturitythetransmittingcells(fig. 11)haveplastidswithplastoglobuli and
onlylittlestarch,smallovalmitochondria0.3-0.5pminlengthwithshortcristae,
dictyosomes with4-5short cisternae(0.3-0.5 pmlength) andsinglestrands ER.
Also somelipid granules and protein bodies witha diameterofabout 1 pmare
present (fig. 12). The observations strongly suggest that the latterare formed
withinER. Thecellsofthetransmitting tissuebecomeloosely organized. Their
middlelamellaeappeardisintegrated. Locally, someelectron-densematerialis
accumulated(fig. II).
Themature micropylarcells(fig. 13) havecellwallswhichare less thick than
the transmitting cell walls. Plasmodesmata are common in all walls. The cell
wallsareinitially homogeneous butatmaturitytheregion ofthemiddlelamellae
as well as the outer wall parts beneath the cuticle bordering the micropyle
DEVELOPMENTOFTHESPINACHOVULE 249
disintegrate. This cuticle is affected too (fig. 13). The large nucleus is located
mostlycentral, withatbothsidesonevacuole.Ovalplastidswithfewthylakoids
and plastoglobuli but without starch are presentin theperipheral cytoplasm,
togetherwithsomemitochondria, dictyosomesand ER. Rarely somesmalllipid
granules are observed.
The chalazalproliferating tissue. The increase ofthe chalazal proliferating
tissue during the latest part ofembryo sac development is mainly due to cell
divisionand partly to cellenlargement. Inthe youngstage (fig. 3) less than 10
cellscanbecountedinafilebetweenthechalazaandtheembryo sac.At maturity
thefilesconsistofover 30cells(fig.4A).Thedivisionactivity isstrongestnearthe
chalaza.Eventually the cells have large vacuoles, a central nucleus with some
cytoplasm and further peripheral cytoplasm (figs. 4E, 15). Plasmodesmataare
commoninallwalls.Thecellswhichare located neartheembryo sac are storing
starch(fig. 14).
The lateraltissue. This consistsof vacuolatecells (fig. 4F), which show con-
siderablevariationin bothsizeandshape.Thelargecentralvacuolesoccupy the
major portion of most ofthecells. These vacuoles contain some ER, dictyo-
somes, mitochondriaandseveralplastids (fig. 16).Intheplastids large amounts
ofstarch accumulateat maturity. Ribosome content is high in the lateralnu-
cellus.Thewalls are thinand havesome pitpairs (fig. 16).
3.3. The female gametophyte
Thedevelopmentofthe femalegametophyte corresponds to the monosporic,8-
nucleatePolygonum type ofMaheshwari. During maturationofthe embryo
sactheareaoftheembryo sacinamedianlongitudinal sectionincreasesabout12
times.Only sporadically a neighbouring nucelluscell becomesresorbed.
At the end ofthe coenocytic period the egg and the antipodal region are
formedfirst by centripetal wall formation.Next, wallsseparate the individual
antipodes and the two synergids. In this youngstage firstly vacuolationand
polarisation takeplace in theegg and centralcell. The egg nucleusand polar
nuclei loose their centralposition. Vacuoles appearrespectively at the micro-
pylar sidein the egg and chalazal in the central cell. All cells show the same
cytoplasmic ultrastructureatthisstageofdevelopment.Thewallsseparating the
variouscellsare thinandshow plasmodesmata. Plasmodesmataareabsentinthe
longitudinal and micropylar walls of the embryo sac. In the chalazal wall,
plasmodesmata are observedfrequently betweenthe chalazalantipodeand the
bordering nucelluscells(fig. 17).
3.4. Histochemistry ofthe mature ovule
Polysaccharides occur intheformofcellwall,cytoplasmic polysaccharide and
starch grains. The reaction of the different parts of the mature ovule after
staining ofallcarbohydrates and separatestainingofstarch canbe seenin table
I.Theoriginalchalazaltissueshows PAS-positive reactionforitscytoplasm. In
theconductivetissueandinthechalazalproliferating tissue mainly thecellwalls
show PAS-reaction. The thickness of their walls is the causeof the different
250 H. J.WILMS
amounts. All cells of the embryo sac are PAS-positive with slight differences
according to their plasm/vacuole ratio and to thethickness ofthe walls with
respective wallprotrusions. Thethincellwallsoftheintegumentscause thefaint
PAS-reaction.Inthemicropylarparts oftheinnerintegument thePAS-reaction
is strongerbecause ofapositive reaction ofthecytoplasm.
Thepresence ofstarch is localized(table /). Red tobrownishred andyellow
colourings areobserved.Theouterintegument gives astrong IKl-reactionwith
anaccumulationintheouterlayer. Thereactionoftheinnerintegument is faint
exceptatthemicropylewhereitis morepositive. Astrongreactionoccurs inthe
nucelluscellsneighbouring theoriginalchalazaltissue,embryo sac andtransmit-
ting part ofthe conductive tissue. The mainpart ofthe chalazal proliferating
tissue coloursyellow.
Table 1. Localization ofreservesubstancesin the differentparts ofthe mature ovule ofSpinacia
oleraceaL.o=faint,+ =positiveand ++ =strongreaction.Ifnoreaction isobservedthesign—
isused.
Integument Nucellus Embryo sac
orig chal condijctive
outer inner ant cc syn egg
chal prol transm microp
Polysaccharides (PAS) 0 0/+ + 0 ++ + ++ + ++ ++
Starch (JKJ) ++ 0 + ye+1l+ow/ ++ - 0 0 + ++
Proteins(Chloramin- 0 ++ + - ++ + 0 ++ 0 ++
T-Schif )
*++ the layer ofthe outercells surroundingthe trace oforiginalchalazal cells shows strong
reaction.
The distributionofproteins is given in tableI. The total innerintegument
shows apositive reactionto protein stainingwithanaccumulationinthemicro-
pylar parts. In the conductive tissue an increase in activity can be observed
towards theembryo sac. Inthe embryo sac the egg and the centralcell show
a strongreaction. Ofallparts oftheovulethe nucleireact positively.
4. DISCUSSION
Goebel(1933) consideredthe ovule of Atriplex hortensisL., belonging to the
family Chenopodiaceae, asamphitropous. Intheamphitropous ovulethe bend-
ingofthenucellusis accompanied by theformation,beneaththeventralface, of
a mass of cells. Thisclassification is based on the cellularorganisation ofthe
mature ovule.On theotherhandBocquet(1959)cameto a differentconclusion
byconsiderating the modeofdevelopmentoftheovuleand theorganisation of
its vasculartissue.He comesto two basic series:the orthotropous seriesandthe
anatropous series. In this view the amphitropous form is considered as an
organisation whichcanappearinbothdevelopmental series;thecentralnucellus
body develops independently. According to the classification of Bocquet the
bitegmic,crassinucellar ovuleofSpinaciaoleraceaL. is ortho-amphitropous. In
thefirst period ofovule development,expansion ofthenucellusisas anextension
251
DEVELOPMENTOFTHE SPINACHOVULE
ofthefunicle, whichis characteristicofthe orthotropous series.Inalaterphase
ofovule development the newly formed chalazal proliferating tissue causes a
cellularorganisation ofthe mature ovuleagreeing withthetermamphitropous.
According to De Boer& Bouman(1972) some outer integuments originate
sub-dermally, the otherouterinteguments and theinner integuments originate
dermally. The initiation and development of the integuments of spinach is
dermaland its finalstructure corresponds with many other dicotyledons (De
Boer& Bouman 1972;Brown& Mogensen1973;Mogensen 1973;Cheah&
Stone 1975). Usually the two cell layers of the inner integument develop dif-
ferently (DeBoer& Bouman 1972;Robertsen 1976).Inspinach, boththerate
of cell divisionand cellenlargement is differentin thetwo layers. During the
development of the integument the plasmodesmata between the two layers
gradually disappear. The merely faint staining for reserve materialsuggests a
limitedsupply andtransportofmetabolites.
Monocotyledons ingeneralshowastrongdividingactivity ofthenucellarcells
betweenthechalazaandthe embryo sac duringthelaterstagesofovuledevelop-
ment (Maze & Bohm 1973;Cave 1975;Berg 1978). Inthe dicotyledons many
taxa have tenuinucellarovules in which the single nucellus layer around the
developing femalegametophyteoftendegenerates beforematurityoftheembryo
sac is reached.Fewovulesofthe studiedtaxa arecrassinucellate.Mostauthors
have not paid attention to a differentiationof the nucellar cells (Coe 1954;
Mogensen1973;Mukkada & Chopra 1973; Norstog 1974;Mitchell 1975).
Morecelltypesaredescribedforcotton (Jensen 1965b) andpartly forPandanus
(Cheah Stone 1975),and Gupta& Rajeswari (1977) have mentionedthe
presence ofnucellar tracheidsin the ovulesof Luffa. Only Malik& Vermani
(1975) suggestasupply route ofmetabolitesto the embryo sac.
In the nucellus of spinach various cell types develop and four specialized
tissuescan be distinguished, although thetissues can not besharply defined.In
our opinionthespecialized charactersofthevarious tissueshavetoberelatedto
variousfunctions.Themicropylarandtransmittingcellsoftheconductivetissue
certainly play a role in relation to the growth of the pollen tube. One can
speculate about a function in attractionand guiding ofthe pollen tube. The
original chalazaltissuelikely servesinthe youngovuleas a directtransporting
pathway fromthechalazato the developing embryo sac. Theseoriginal chalazal
cellshavesimilarcharacteristicsasthe transfercellsofGunning& Pate(1969),
especially thestrongly developed ER system andthemanyplasmodesmata inthe
shorttransfer walls. Up to maturity the chalazal proliferating tissue develops
many files and causes a structural cut off ofthedirect transport pathway of
metabolitesfrom the funicularvascular strand to the original chalazal tissue.
The following supply ofreserve metabolitesis mostly stored as starch in the
chalazal proliferating and lateralcells which are located around the original
chalazaltissue. At maturityoftheovuleand probably also during the develop-
mentoftheembryo andendosperm, theoriginalchalazalcellspossibly function
as sink and transfer of nutrients, passed from the chalaza by the chalazal
proliferating tissue.
252 H. J. WILMS
The possible pathway of polysaccharides in spinach is proposed for three
different phases of development {diagram 1). The young stage represents the
developingovule.Inthematurestagetheembryo sacisfunctionaland thefileof
chalazal proliferating cells are formed. In the fertilized stage the zygote and
endosperm starttheirdevelopment.
Diagram 1. Theproposedpathwayofpolysaccharides inthespinachovuleduringthree stagesof
development.A, youngstage;B. maturestage andC. afterfertilization has takenplace.
During successive stages of development and growth ofthe ovule there is
substantialpolysaccharide storing. Inthe youngstagepolysaccharides are sto-
red in the file of nucellus cells beginning at the chalaza and ending at the
micropyle. A concentrationofstoragematerial, starch grains,isobservedinthe
transmitting cells ofthe conductive tissue. The file of original chalazal cells
shows astrong PAS-positive cytoplasmic reaction. Nearmaturity metabolites
are storedinthe innerand outer integuments. Withthe process offertilization
insolublepolysaccharides are gradually depleted fromthe nucellus.Theintegu-
ments and many nucellar cells are storage reservoirs for the main source of
energyneededforsubsequent embryo andendosperm development. Thepresent
data indicate that during ovular development the nucellus has only a minor
nutritiverolein relationto the relativesmall embryo sac.
The presence ofprotein inseveral parts ofthe mature ovule seems strongly
related to thereproductive functionofthe femalegametophyte. The positive to
strong reactionin the tissuesofthemicropyle surrounding innerinteguments
and the conductive tissue of the nucellus can be related to pollen tubepenet-
ration. The presence ofproteins in eggand centralcellcan be related to their
futurefunction.Theobservationsin Zephyranthus ofMalik& Vermani(1975)
indicatethe samedirection.They foundinthe youngantipodals a highamount
ofproteins, whereasat the approachoffertilizationembryo sacs containlow
amounts of proteins, mainly concentrated in the centralcell and in the egg.