The unusual tegumental tissues of the Lunaria annua (Brassicaceae) seed: a developmental study using light and electron microscopy

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ContentslistsavailableatSciVerseScienceDirect

Flora

j o ur na l h o m e p a g e :w w w . e l s e v i e r . d e / f l o r a

The

unusual

tegumental

tissues

of

the

Lunaria

annua

(Brassicaceae)

seed:

A

developmental

study

using

light

and

electron

microscopy

Stefano

Mosti

a

_,

_Cynthia

_Ross

_Friedman

b

_,

_Fabio

_Piccolin

a

_,

_Pietro

_Di

_Falco

a

_,

_Alessio

_Papini

a,∗

a_Department_of_Evolutionary_Biology,_Università_di_Firenze,_Via_La_Pira,_4,₅₀₁₂₁_Firenze,_Italy

b_Department_of_Biological_Sciences,_Thompson_Rivers_University,₉₀₀_McGill_Road,_Kamloops,_British_Columbia,_Canada_V2C_0C8

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received23May2012 Accepted10September2012 Keywords: Brassicaceae Lipids Seeddispersal Seedinteguments Seedanatomy Buoyancy

a

b

s

t

r

a

c

t

Withlight,fluorescence,transmissionelectron,andenvironmentalscanningelectronmicroscopywe studiedthedevelopmentoftheLunariaannuaL.(Brassicaceae)seedsinordertorevealbasicanatomical informationabouttheunusualtissuesoftheseseeds.Inparticulartheseedtegumenttissuespossess complexmorphologicalaspectsthatarerelevanttothebiologyandecologyofthisplant.A sclerenchy-matictissueastheinnermostlayerofthetegumentsapparentlyoffersrobustprotectionfortheembryo, yetisorganizedtobeflexible.Thistissuelikelycontrolsthepassageofwaterfromthetegumentallayers towardstheembryo.Wereportherethepresenceoftanninsinthepre-sclerenchymaticlayerofthe unripeseed.Theinnertegumentalsohousesaspongytissuewithwideintercellularspaces.Thistissue couldimpartbuoyancytotheseeds,whichpossiblymightberequiredforwatertransport.Thestructural featurescouldindicatethatLunariamayhaveevolvedinaMediterraneanenvironment,whichis charac-terizedbyalongdryseason,butwithalargeamountofrainfallconcentratedinshortperiods.Probably, notonlythetypicalenlargedandflattenedfruitsofLunariacaneasilyfloatandbedispersedawayfrom themotherplant,butalsotheseedshavethisdispersalpeculiarityafterreleasefromthesilicules.

Introduction

LunariaL.isagenusofBrassicaceaecharacterizeda bylarge membranous silicula which is oblong-elliptical tosuborbicular, roundedatthebaseandapexandcontainingdisc-shapedseeds (Fig.1;seealsoBall,1993).LunariaannuaL.isaspecies originat-ingfromwesternAsiaandsouth-easternEurope(Pignatti,1982), andhasnownaturalisedinmanytemperatecountries,whereitisa popularornamentalﬂower(Guoetal.,2009).Abiennialor peren-nialcruciferousplant,ithasahigh(35%)oilcontentinitsseeds. Theoil,comprisedofverylong-chainmono-unsaturatedfattyacids (e.g.,nervonicacid)hasbeenusedasanindustriallubricant(Guo etal.,2009;MastebroekandMarvin,2000)andmayhavepotential interestforotherapplications,e.g.forbiofuelproductionlikeother Brassicaceae(Appelquist,1976;Ciccarellietal.,2010;Papiniand Simeone,2010;Papinietal.,2010a,b).

WhentheseedofL.annuaisripe,thelipidsareprimarilystored withintheembryo,whichisseparatedfromtheoutside environ-mentbytheseedcoat(testa),whichiscomprisedoftegumental layers(canalsobecalled‘integuments’).Onlyalimitednumber ofstudiesdescribethesixtegumentaltissuestypicalforL.annua seeds.Threeexternallypositionedlayersofthedevelopingseed

∗ Correspondingauthor.Tel.:+390552757395.

E-mailaddress:alpapini@uniﬁ.it(A.Papini).

derivefromtheouterovuletegument,whilethethreemore inter-nallayersoriginatefromtheinnertegument(Bouman,1975).The mostexternaltissueistheepidermis,thesecondthesub-epidermal tissue,whilethethirdhasbeendefinedas‘palisadetissue’(Van Caeseeleetal.,1982;VaughanandWhitehouse,1971).Thefourth, fifthandsixthtissuelayersoftheL.annuatestacorrespond, respec-tively,tothesuperficial,middle(or‘spongy’),andbasaltissuesof theinnertegument(Bouman,1975).Themiddleandbasaltissues are particularlystriking due totheirdarkappearance.Vaughan andWhitehouse(1971)calledthebasaltissue‘thepigmented tis-sue’becauseofitsstrongreactivitytoToluidinebluestain.Inthe ripeseed,cellsofthebasaltissuearedead,andwereidentifiedas sclereidsbyBouman(1975).Inourstudy,weundertookan investi-gationoftheanatomyandultrastructureofthetegumentaltissues inL.annuathroughouttheembryodevelopmentuntilseed ripen-ingwasachieved.Theultrastructuralobservationsofthesetissues arethefirstoftheirkindforL.annua.Wealsoexaminedthe cyto-chemistryofthesetissuesatmaturityinordertobetterunderstand thenatureoftheseimportanttissues.

Materialsandmethods

Plants

LunariaannuaplantsweregrownintheBotanicalGardenofthe UniversityofFlorence(Italy).Unripeandlaterripefruits(thetypical

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FLORA-50645; No.ofPages10

2 S.Mostietal./Floraxxx(2012)xxx–xxx

Fig.1. AmaturefruitofLunariaannuaL.(silicula)fromanHerbariumsample

col-lectedatCertosa(nearFlorence).

enlargedandﬂattenedsiliquesofLunaria)werecollectedat differ-entdevelopmentalstagesrecognizedonthebasisofthesilicule dimensions:3.5cm× 2.5cm;4cm× 2.6cm;4.5cm× 2.9cm(ripe) andtheseedsusedfortheinvestigations.Someoftheripeseeds werehydratedfor24h.

Embeddingprocessforlightandelectronmicroscopy

Seeds at different stages of development were preﬁxed overnightin1.25%glutaraldehydeat4◦Cin0.1Mphosphatebuffer (pH6.8),andthenﬁxedin1%OsO4inthesamebufferfor1h.After

dehydrationinanethanolseriesandapropyleneoxidestep,the sampleswereembeddedinSpurr’sepoxyresin(Spurr,1969).

Sectioningandstainingforlightandﬂuorescencemicroscopy

SeedsembeddedinSpurr’s epoxyresinweresectionedwith glassknivestoobtainsemi thinsections(1–5␮m),whichwere stainedwithToluidineblue,0.1%,thenobservedandphotographed withaLeitzDMRBlightmicroscope.Seedsthatwerenotembedded wereinsteadsectionedwithaCryostattogeneratesemi-thin sec-tions(10–20␮m).Someoftheseseedsectionswerestainedwith 1%phloroglucinol(w/v)in12%HClfor5minandobservedwitha brightfieldlightmicroscopeforthedetectionoflignin(Fernandez andHeath,1986).Anothersetofcryostatsectionswerestained withSudan IIIfor thedetectionand localizationoflipidsunder brightfieldmicroscopy(Brundrettetal.,1991).Theremainderof theCryostatsectionswerestainedwithFluorol Yellow088and viewedwithafluorescentmicroscopeLeicaDMRBFluointherange of515–565nm(green) todetectlipids(Brundrettet al., 1991). Seriesofimagesweretreatedwiththepythonprogram ALLAM-ODA2.0(Papini,2012)toreducenoise.

Transmissionelectronmicroscopy(TEM)

PiecesofseedsembeddedinSpurr’sepoxyresin(Spurr,1969) werealsocutwithadiamondknifetogeneratesectionsthatwere approximately80nmthick.Thesewerestainedwithuranylacetate (GibbonsandGrimstone,1960)andleadcitrate(Reynolds,1963), andthenexaminedwithaPhilipsEM300TEMoperatingat80kV.

EnvironmentalScanningElectronMicroscopy(ESEM)

SeedsofL.annuawerehand-cutwitharazorblade;resulting sections(0.5–1mm)weremountedonstubs.Weuseda Quanta 200EnvironmentalScanningElectronMicroscopy(ESEM,Fei Cor-poration,TheNetherlands)operating inlow-vacuummode(the

chamberpressurewaskeptat1Torr)toobservethesamplesand generateimagesbytheCEMEcenter(www.ceme.ﬁ.cnr.it).

Results

Thetegumentallayersoftheseedtestabecomeclearlyevident asthesixdistincttissues(epidermal,sub-epidermaltissue,and pal-isadeoftheoutertegument;superﬁcial,spongy,andbasalofthe inner)onlywhentheembryohasprogressedintotheearlyheart stage(Fig.2B).Seedsatthisstagepossessafree-nuclearendosperm withalargecentralvacuoleandremnantnucellus(Fig.2A). Follow-ingisadescriptionofthetissueregionsofthetegumentallayers surroundingaseedwithaheartstageembryo,withanemphasis ontheultrastructure.Thetissuesaredescribedfromtheoutside towardstheinternalpartoftheseed(occupiedbythecavity hous-ingtheendospermandthedevelopingembryo).

Tegumentaltissuesattheheartstage

Epidermaltissue(outertegument)

Theepidermaltissue,themostexternaltissue,ismonolayered andcomposedofcellswitharoundishshape(Fig.2B).Thesecells havethickexternaltangentialwallswithanoteworthycutinlayer, identiﬁedwithTEMtobeastronglyelectron-densecoat(Fig.2C). Thenucleiarelocatedattheperipheryofthecells(Fig.2D). Rela-tivelylargevacuolesoccupyacentralposition,whileothersmaller vacuolesalongwithlargeglobularstarchcontainingamyloplasts couldbeobservedintheperipheralcytoplasm(Fig.2C).

Sub-epidermaltissue(outertegument)

The sub-epidermal tissue shows two to many cell layers (Fig.2B).Thecellsaresmallerandmoreﬂattenedthanthoseof theepidermallayer.Thevacuolesinsub-epidermalcells occupy asmallerpartofthecellvolumeascomparedwithcellsinthe epidermallayer(Fig.2E).Thesesub-epidermalcellsalsocontain prominent amyloplastswithstarch (Fig. 2E).A main featureof thesub-epidermalcellcytoplasmisthepresence oflongrough endoplasmicreticulum(RER)cisternae.ThisRERseemstobe con-tinuouswithlargevesiclesthatcontaindenselygranularmaterials ofmediumelectrondensity,andthemembranesofthesevesicles appeartobecoveredwithribosomes(Fig.2F).

Bundlesofconductivetissuearepresentinthesub-epidermal region. Conductive elements (tracheid-like cells with annular thickenings)arealsovisiblein islandsoftissuesobservedclose tothehilum-micropylarzoneoftheseed(Fig.3A).Cellsofthese islandsaresmaller,haveamoreelectron-densecytoplasm,andare lessvacuolatedthanthesurroundingcells.

Palisadelayer(outertegument)

Thepalisadelayerisprimarilyamonolayeredtissue,andforms thebasallayeroftheoutertegument(Fig.2B).Thecellscomposing thistissue arenormallyradially elongatedand possessstrongly thickenedinner tangential and basal radial walls(Fig.3B).The nucleus ofthesecells is spherical andmainly euchromatic,but containssomecondensedheterochromatin.Asinglenucleolusis typicallyvisible(Fig.3B).Thecytoplasmicmatrixhasamedium electron density, with ribosomes either free or associated in polysomes(Fig.3C),andtypicallycontainsoneortwolarge vac-uolesandsomesmallervacuoles(Fig.3B).Matureordeveloping amyloplastsare alsopresent in the cytoplasm,each containing onetotwostarchgrains(Fig.3B).Someamyloplastsseemtobe engulfed in vacuoles (Fig. 3B). Close to thewalls, dictyosomes producevesicles,someofwhichcanbeseenfusingwiththeplasma membrane(Fig.3C).Structureswithaparacrystallineappearance arefrequentlyobserved (Fig.3D). Whenviewedinlongitudinal

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Fig.2.(A)SectionofaL.annuaunripeseedwiththeembryoatincipientearlyheartstage.Thetegumentallayersofthetesta(T)arenotfullydeﬁned.Thefuniculus(Fu),

endosperm(En)withlargecentralvacuole(V),andremnantnucellus(Nc)areevident.(B)SectionofaL.annuaunripeseedwiththeembryoatearlyheartstage.Sixdifferent

typesoftissueareevident:Epidermaltissue(Ep),Sub-epidermaltissue(SEp),Palisadetissue(Pa),Superﬁcialtissue(Su),Spongytissue(Sp),Pre-sclerenchymatictissue(Psc).

(C)Epidermalcell.Alargevacuoleoccupiesalmosttheentirecellvolume.Intheremainingcytoplasm,smallvacuolesandlargeamyloplastsareclearlyvisible.Thetangential

externalwallisthick,withanoteworthyelectron-densecutinlayer(arrow).(D)Epidermalcell.Theﬂattenednucleuscontainingaprominentnucleolusisattheperiphery

ofthecells.Thethickexternaltangentialwall,withastronglyelectron-densecoatofcutinisalsoclearlyvisible.(E)Sub-epidermalcell.Flocculentmaterialisvisibleinthe

bigvacuole.RERcisternaeandvesicleswithamediumelectrondensity(arrows)contentarevisible.(F)Portionofsub-epidermalcell.TheRERcisternaearecontinuous

withvesiclesthathaveadenselygranularcontentandseemtobecoveredwithribosomes(arrows)(Keytolabellingofﬁgures:LightMicroscope:Al:Aleuronelayer;Em:

Embryo;Pa:Palisadelayer/tissue;PSc:Pre-sclerenchymaticlayer/tissue;Sc:Sclerenchymaticlayer/tissue;T:testa;TEM:TransmissionElectronMicroscope;d:dictyosome;

ld:lipiddroplets;m:mitochondria;N:nucleus;nl:nucleolus;pc:paracrystallinebody;Pl:plastid;RER:roughendoplasmicreticulum;SER:smoothendoplasmicreticulum;

S:starch;V:vacuole;W:wall).

section these paracrystalline bodies take onthe appearance of ﬁlamentbundles.Individualﬁlamentsare30nmindiameter.

Superﬁcialtissue(innertegument)

Thistissueformstheoutermostcoveringoftheinnertegument, andisaonetothree-cell-layeredtissuefoundbetweenthepalisade layeroftheoutertegumentandthespongytissueoftheinner tegu-ment(Fig.2B).Thecellsconstitutingthetissuedifferinbothshape anddimensionandcouldbeisodiametric,radially elongated,or tangentiallyelongated(Fig.3E).Thenumberoflayersisvariable

aswell,rangingfromone(themostfrequentcondition)totwoor three(Figs.2Band3E).Sometimeseventhesinglecelllayerwas incomplete.Thecellsinthistissuehavelargevacuolesaswellas somesmalleronescontaininggranularmatterofmediumelectron densityandamyloplasts(Fig.3F).

Spongytissue(innertegument)

Thisis the ﬁfthtissue from theoutside toward theinternal chamber,andisamultilayeredtissue.Itshowsdifferent morpholo-giesdependingonthepointofobservation:inthelateralareaof

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Fig.3.(A)SectionofaL.annuaunripeseedwithanembryoattheearlyheartstage;sectionfeaturesthesub-epidermaltissue.Conductiveelements(arrows)inthe

hilum-micropylarzoneoftheseedareevident.(B)Palisadelayer.Thecellsareradiallyelongated.Thetangentialinternalwallsandthebasalradialwallsarestronglythickened.

Amyloplastscontainingstarchcanbeseenwithinthevacuoles.(C)Palisadelayer.Inthisportionofthecell,adictyosomeproducingvesiclesisvisible.Thesevesiclesappear

tofusewiththeplasmamembrane.Inthecytoplasm,manypolysomes(arrows)areevident.(D)Palisadelayer.Paracrystallinestructures(pc)arefrequentlyobserved.In

longitudinalsection,thesestructureslooklikeﬁlamentbundles(arrow).(E)DetailofasectionofaL.annuaunripeseedwithanembryoattheearlyheartstage.Thewidth,

numberoflayers,andshapeofthesuperﬁcialandspongytissuesvariesdependingontheareaoftheseed.Conductiveelementsinthesub-epidermaltissuearevisible

(arrows).(F)Superﬁcialtissue.Thecellshaveonelargevacuole,andsomesmalleronescontaininggranularmaterialofmediumelectrondensity(Keytolabellingofﬁgures:

seelegendofFig.2).

theseed,thistissueappearsquitecompactwithreduced intercel-lularspaces(Fig.2B).Thecellsinthisareaappeartoberoundish, ovoid,orquiteelongated.Conversely,inthedorsalareaoftheseed, thevolumeofthistissueincreasesremarkably,andthe intercellu-larspacesappearasbeingdilated(Fig.2B).Thecellsofthedorsal areaareelongatedoratleastelliptic (Fig.4A).Whiletheentire spongytissueismultilayered,thenumberoflayerswasdifﬁcultto determine,particularlyinthedorsalarea,wheretheintercellular spacesareenlarged.Wherethecellsremainincontactwitheach other,plasmodesmataareevidentattheadhesionpoints(Fig.4B). Ultrastructurally,thecellsofthistissuearesimilar.Theytypically possessamain(central)vacuolewithanelectron-transparent con-tentaswellassmallervacuoles(Fig.4A).Thenucleusmostoften

hasaperipheralpositionandaﬂattenedorirregularshape.Itis typicallyalmostcompletelyeuchromaticwithaprominentsingle nucleolus(Fig.4C).Theremainingcytoplasmpossessesapoorly electron-densematrixand relativelylargeamyloplaststogether withlongRERcisternaedisposedinparallelarrays(Fig.4BandD). Thewallshaveaﬁbrillarappearance(Fig.4D).

Basalor‘pre-sclerenchymatic’tissue(innertegument)

Thebasaltissueisthemostinternaltissueamongthoseforming thetegumentsofL.annua.Asitlaterbecomesscleriﬁed,wehave decidedtocallthistissuethe‘pre-sclerenchymatic’tissuewhenit isobservedatthisstageofdevelopment.Thepre-sclerenchymatic tissuecoverstheinternalchamberwheretheembryowilldevelop,

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Fig.4. (A)Cellinthespongytissue(dorsalareaofseed)possessingatypicalelongated-ellipticshape.Onemainvacuoletogetherwithsmallervacuolesandsomerelatively

largestarchgrainsarevisible.(B)Cellsofthespongytissue.Wherethecellsstayincontactwitheachother,plasmodesmatatraversingthewallsintheadhesionpoints

canbeseen(arrows).(C)Anareawherecellsofthespongytissueremainincontact.Aﬂattenednucleusinaperipheralpositionisevident.Thisnucleusispredominantly

euchromaticwithaprominentnucleolus.(D)Anotherareawherecellsofthespongytissueremainincontact.Thewallshaveaﬁbrillarappearance,andinthecytoplasm,

theRERcisternaearedisposedinparallelarrays.(E)Cellsofthepre-sclerenchymatictissue.Bigvacuoleswithstronglyelectrondensedepositsclosetothetonoplastform

clustersthatoccupythemainpartofthecellvolume.Granularelectron-densematerial(arrows)ispresentintheradialandexternaltangentialwalls.(F)Cellsofthe

pre-sclerenchymatictissue.Thenucleusisroundwithaprominentnucleolus,andinthecytoplasm,plastidsapparentlydevelopingtoamyloplastsarepresent.(G)Cytoplasmof

cellsinthepre-sclerenchymatictissue.Masses(arrows)ofelectron-densegranules(unboundbyanymembrane;i.e.,‘free’)arefoundclosetoactivedictyosomes.Sometimes,

suchmasseswereobservedinthevicinityofthebigvacuoletonoplasts(Keytolabellingofﬁgures:seelegendofFig.2).

and is in direct contact with the endosperm; cells in contact withtheendospermhaveconvexwalls.Thetissueappearstobe constitutedbyonecelllayeratthemicropylarpole,andmultiple celllayersatthechalazalpole(Figs.2Band3E).

Ultrastructurally,bigvacuolesinclustersoccupythemainpart ofthecellularvolume(Fig.4E).Withinthesevacuoles,appressedto thetonoplast,continuousdepositsofstronglyelectron-denseand compactmaterialareevident(Fig.4E).Sometimesthesedeposits extendtowards thecenter of thevacuoles, but as a more rar-eﬁed and apparentlyﬂoccular material(Fig.4E and F). Smaller vacuolesoccupyamoreperipheralposition,areweakly electron-dense, and lack the electron-densedeposits seen in the larger

vacuoles(Fig.4E).Massesofelectron-densegranulescanbeseen freein thecytoplasm,closetoactivedictyosomesthat are pro-ducingvesicles(Fig.4G).Theseelectron-densecytoplasmicmasses canalsobeseeninthevicinityofthetonoplastof thebig vac-uolesthatcontaintheelectron-densematerial(Fig.4G).Granular electron-densematerialisalsopresentoutsidetheplasma mem-brane in theareas of the radial and external tangential walls, andcanbeseeninthewallsthemselves,apparentlywithinthe middle lamella(Fig.4E).Nucleiaretypicallyroundand contain aprominentnucleolus; thecytoplasmpossessesa densematrix whereplastids(apparentlydevelopingintoamyloplasts)are evi-dent(Fig.4F).

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Fig.5. (A)Detailofapre-sclerenchymaticcelllaterindevelopment.AlargequantityofSERwithswollencisternaeaswellasactivedictyosomesproducingvesicleshave

appeared.Theelectron-densematerial(arrows)intheradialandoutertangentialwallshasbecomeabundant.(B)Apre-sclerenchymaticcellverylateindevelopment,prior

toscleriﬁcation.Thecytoplasmhasbecomestrongly-electrondense.Somevacuolescouldbestillrecognizedtogetherwithactivedictyosomescontinuingtoproducevesicles.

(C)SectionofaL.annuamatureseedfeaturingthecellsoftheepidermallayerandsub-epidermallayer,whichhavedied(cellsappearempty).Thecellwallspersist,anda

bandappressedtotheoutertangentialwallsoftheepidermallayerisstronglyToluidineblue-positive(pink).(D)ImageshowingdetailofthewingofaL.annuamatureseed,

andfeaturingthematurepalisadelayer.Thesectionhasbeenstainedwithphloroglucinol.Thewallsofthepalisadelayercells,whichhavedied,arephloroglucinol-positive

(red).(E)Cellsofthespongytissueinadryripeseed.Thecellshavedied.Theprimarywallsappearelectron-denseandthesecondarywallsconstitutethemajorityofthecell’s

volume.Theinternallumencontainsosmiophilicmaterialandvacuoles(arrow).(F)Cellsofthespongytissueinthedryripeseed.Intheintercellularspaces,electron-dense

lipidicdroplets(arrows)arevisible.(G)Cellsofthespongytissueinthehydratedripeseed.Thesmallerlumen(arrows)appearselectron-transparentorcontainsscarce

amountsofﬁbrillarmaterial(Keytolabellingofﬁgures:seelegendofFig.2).(ForinterpretationofthereferencestocolorinFigurelegend,thereaderisreferredtotheweb

versionofthearticle.)

As development progresses, the cells belonging to the pre-sclerenchymatictissuebecomemoreelectron-dense,andalarge quantityofSERwithswollencisternaeappears(Fig.5A).The dic-tyosomesbecomeveryactive,producingsmallvesicles(Fig.5A). The electron densematerial in the radial and outer tangential wallsincreasesinvolume(Fig.5A).Finally,thecytoplasmofthe pre-sclerenchymaticcellsbecomesstronglyelectron-dense, oblit-eratingmuchofthecellularultrastructure(Fig.5B).Nevertheless, somevacuolescouldstillberecognizedtogetherwithactive dic-tyosomescontinuingtoproducevesicles.

TissuesinthetegumentsoftheripeseedofL.annua(all tissuesarenowdead)

Outertegument

Inthematureseed,cellsoftheepidermallayerhavedied,as thecells appearempty.Thecell wallspersist, though,withthe outertangentialonesbeingparticularlythick:abandappressed totheoutertangentialwallsis stronglyToluidine blue-positive (pink)(Fig.5C).Liketheepidermallayer,theonetothreelayersof thesub-epidermaltissuearealsodeadatmaturity(Fig.5C).These

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cellsremainirregularlyshaped,aresomewhatﬂattened,andhave slightly thickenedwalls.Thebasal layeroftheinner tegument, thepalisadelayer,nowformsacontinuousband(Fig.5C):these cellshavediedaswell.Theyhaveaprismaticshape,andpossess thickened,phloroglucinol-positive(thuslignin-containing)walls, particularlyintheareaswheretheseedhasformedoutgrowthsor ‘wings’(Fig.5D).

Innertegument

Atmaturity,thesuperﬁcialtissue,whichwastheoutermost tis-sueoftheinner (in)tegument,isnolongerdistinguishable asa discretetissue,asithasapparentlymergedwiththespongytissue. Cellsofthespongylayerremainlooselyconnectedtoeachother andpossesslargeintercellularspaces(Fig.5C).Theultrastructureof thesecellsindicatesthattheytooaredeadatmaturity,constituted mainlyofwallmaterial(Fig.5E).Theprimarywallsare electron-denseandformedbycompactmaterial,whilethesecondarywalls comprisethemajorityofthecellularvolume.Thesesecondarycell wallsarecomposedoflooseﬁbrillarmaterialamongwhichsome electron-densegranularmaterialisvisible(Fig.5EandF). Electron-densedroplets,likelycomprisedoflipids,aresometimesevident withinthewideintercellularspaces(Fig.5F).

Whilecellwallmaterialﬁllsthemajorityofagivencell’svolume inthespongytissue,thesecellsstillpossessaninternallumen.In dryseeds,thelumenisapparentlyoccupiedbyosmiophilic mate-rialwithembeddedvacuoles(Fig.5E).Inhydratedseeds,however, thelumenissmallerthaninthedryseed,andissometimesnot evenvisible.Thelumenappearseitherfullyelectron-transparent ormaycontainscarceamountsofﬁbrillarmaterial,whichisfound predominantlyalongthewalls(Fig.5G).

WhenstainedwithFluorolYellow(Fig.6A),thespongytissueof thematuredryseedshowsonlycondensedclustersoflipidgrains liningthewalls,andhasnostainingreactionatallwhentreated withSudanIII(Fig.6B).Onthecontrary,thespongytissueofthe hydratedseedstainedwithFluorolYellow(Fig.6C),andSudanIII (Fig.6D)becomesdiffuselypositiveforthepresenceoflipids,as doestheembryoandthealeuronelayer.

At maturity, the basalmost tissue, formerly the pre-sclerenchymatic tissue, has become fully sclerified, and can nowbecalledthe‘sclerenchymatictissue’(Fig.5C).Itscellsshare a boundarywith thealeurone layerof theendosperm.Cells of thesclerenchymatictissuehaveverythickenedradialandinner tangentialwalls,givingthesecellsa‘U’-shapedconfigurationin whichtheylackanobviousoutertangentialwall.Thesecellsare deadatmaturitybutnotempty,sinceanamorphousandcompact densematerialisvisible(Fig.5C).TheESEMimages(Fig.6E)show thatthematerialinthesedeadcellscomprisessolidblocksofmore orlessregularprismaticshape(Figs.5Cand6E).Thesesolidblocks appeartobeformedbyappositionofbricklikedeposits(Fig.6F). Smallspherical electron-dense massesare often evident inthe cavity betweenthe sclerenchymaticcell wallsand theinternal blocks ofthesecells (Fig.6F). Boththecellwalls and thesolid internalblocks ofthesclerenchymaticlayerare phloroglucinol-positiveforlignins,withtheblocksstainingparticularlyintensely (Fig.6G).Thebasesoftheinnertangentialwallsincontactwiththe aleuronelayershowawidespreadporosity;themostthickenedof thesewallsappearstratified(Fig.6H).

Discussion

Developmentintheoutertegumentaltissues

Epidermallayerandsub-epidermaltissue

Oneoftheobviousfeaturesofthedevelopingepidermallayer inL.annuawasthecopiousamountofamyloplasts.Thepresence

ofstarchgranulesinexternalseedlayershasbeenlinkedtothe formation of mucilages (Harris, 1991), hydrophilic polysaccha-rides producedin speciﬁctissuesof seedsinvarious speciesof angiosperms(Westernetal.,2000)andparticularlyinBrassicaceae (VaughanandWhitehouse,1971).However,wedidnotobserve any mucilage production by the epidermal layer of L. annua norpre-secretorycytoplasmicmucilage storageasdescribedby Windsoretal.(2000)forArabidopsis.Thestarchmaybeinstead usedinthethickeningoftheepidermalcellwalls.

The sub-epidermal tissue possessed cells also containing numerousandlargeamyloplasts,againlikelytobeusedinwall thickening.Thetracheid-likeconductiveelementspresentinthe conductivetissuebundlesofthistissuemaybetracheoids, vascu-larelementscommonlyfoundattheendoftheleafveins(Gonzalez andLopez,2010)anddocumentedinseedswingsofBignoniaceae (Lerstenet al., 2002).Lunaria is theonly genusof Brassicaceae known to have an extensive tegumentary seed vascularization (Bouman,1975).Inleaves,thesetracheoidsfunctioninwater stor-age, air storage, and mechanical support (Gonzalez and Lopez, 2010), and in the seed, theymay have roles in short distance watertransport(Fahn,1990).Ultrastructurally,themost interest-ingaspectofthesub-epidermaltissuewasthepresenceoflong RER tracts ending in vacuoles ofmedium electron density.The contentsoftheseRERcisternaeandassociatedvacuoleswere sim-ilarlygranularandelectron-dense,probablyofaproteinicnature. Suchproteinsmaybeenzymesinvolvedinthedevelopmental pro-cessesandintheautophagicactivityinthis tissueandpossibly adjacenttissues,particularlythepalisadetissue(seenextsection).

Palisadelayer

In thepalisade layer,thedictyosomesand secretoryvesicles werelikelyfunctioningtotransportcelluloseandpecticsubstances necessaryforthewallthickeningprocesses,assuggestedalsoby VanCaeseeleetal.(1982)inBrassicacampestris.Thepresenceof starchgranuleswouldrepresentacarbohydratesourceforthese processes.

Theparacrystallinestructuresobservedinthecellsofthistissue aredifficulttopositivelyidentifywithoutimmunocytochemistry, butcouldbeperoxisomesorglyoxysomes,organellesthatplaya role in breakdownof verylong chainfattyacids through beta-oxidation (Michelsetal.,2005).Van Caeseeleet al.(1982) saw similarstructuresinB.campestris,callingthem‘tubular paracrys-talline structures’,but didnot attribute any particularfunction to them. However, when viewed in longitudinal section, these paracrystallinestructurestookontheappearanceoffilament bun-dles. What we putatively call ‘peroxisomes’ might be filament bundlesincrosssection.Iftheparacrystallinebodiesaretrulythe filamentbundles,theymightbenecessarytogivemorerigidityto thesecellswhiletheyarestillalive.Thediameterofthesefilaments (30nm)correspondsmorecloselytothedimensionofmicrotubules (about25nm),butwithouttheirtypicalmorphology.

Thepresence ofamyloplasts withinthevacuoles ofpalisade layer cells signals the onset of autophagyand hence the later developmentalstages,aswassimilarlyobservedinB.campestris (VanCaeseeleetal.,1982).Theautophagic processesare proba-blypreliminarytoafinalprogrammedcelldeath(PCD)ofthese cellsatmaturity.InBrassicanapus,thepalisadelayeristhemain supportingtissueinthematureseed(Iwanoskaetal.,1994).The deadcells ofthepalisadelayerremaininginthematureseedof L.annuado notappear tohavethesameimportant supporting function,exceptinthe“wing”zone,wherethewallsappeared lig-nified(phloroglucinol-positive).Theselignifiedregionsmightalso functiontoblockwaterabsorption,enablingseedbuoyancy.Other lignifiedtissuesinseeds,suchasthesclerenchymaticparenchyma belowtheepidermisinAcacia(Venieretal.,2012),arebelieved

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Fig.6.(A)Lightmicroscope(ﬂuorescence)imageofasectionfromadrymatureseedstainedwithFluorolYellow.Onlysomeclustersoflipidgrains(arrows)inthespongy

tissueareﬂuorescent.(B)SectionofadrymatureseedstainedwithSudanIII.ThespongytissueinthedrymatureseedisnegativefortheSudanIIIstainwhiletheembryo

containesmanySudanIII-positivedroplets.(C)Lightmicroscope(ﬂuorescence)imageofasectionfromahydratedmatureseed,stainedwithFluorolYellow.Thetissuehas

becomediffuselyﬂuorescent,ashasbecomeaswelltheembryoandthealeuronelayer.(D)SectionofahydratedmatureseedstainedwithSudanIII.Thespongytissuehas

becomediffuselypositiveforSudanIII.(E)ESEMimage.Thecellsofthesclerenchymaticlayerofthedryripeseedhaveverythickenedradialandinnertangentialwalls,

givingthesecellsatypical‘U’-shapedconﬁguration.Thecellularcontentisrepresentedbyasolidblock(asterisk)ofmoreorlessregularprismaticshape.(F)Detailofcells

ofthesclerenchymaticlayerintheripedryseed.Thesolidblockswithinthesedeadcellsappeartobeformedbyappositionofbrick-likedeposits(asterisks).(G)Image

ofasectionofadryripeseedstainedwithphloroglucinol.Theinternalblocks(asterisks)ofthesclerenchymatictissueareintenselyphloroglucinol-positive.(H)Detailof

sclerenchymaticcellsinthedryripeseed.Thebasesoftheinnertangentialwallsincontactwiththealeuroneshowawidespreadporosity(arrows)(Keytolabellingofﬁgures:

seelegendofFig.2).

tocontrolthewaterﬂuxfromoutsidetheseed.Inthiscase,such controlcouldalsogovernseeddormancy.

Developmentintheinnertegumentaltissues

Superﬁcialandspongytissues

Thesuperﬁcialandspongytissuelayers,strictlyinterconnected tooneanother,mergeintoonetissueatseedmaturity.Theincrease inseeddimensionoccursfollowingboththedilationoftheembryo chamberandthegrowthofthespongytissue,withsomeofthe

volumeincreaseattributabletotheenlargementofthe intercellu-larspaces.Thesymplasticcontactsareneverthelessmaintainedas longasthecellsremainviable,probablytomaintaindevelopmental synchronicity.

Asthespongytissueapproachesmaturity,thewallsdistendand thecentralvacuoleineachcellincreasesinvolume.Thenumerous smallervacuolesthatalsoappearareprobablyrelatedtoautolytic processes,whichareparticularlyactiveagainsttheamyloplasts. Therefore,thestarchinamylopastsoftheyoungerspongytissueis likelyneededtoproviderawmaterialforthewallexpansion.The

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manyribosomes,bothfreeandassociatedwithintheER,arelikely involvedinproducinglyticenzymes.Thedeadcellsoftenshowed tracesofpartiallylysed,osmiophiliccytoplasmandnuclei, hall-marksofPCDinplants(Brighignaetal.,2006;Papinietal.,1999, 2010c,2011).Atmaturity,thesedeadcells arecharacterizedby theirwalls,whichpossessalooseandﬁlamentousmatrix.While poroustissuesarerareinplants,Cycasseedspossessaspongy tis-suethatinitiallyimparts buoyancytotheseedstoenabletheir dispersal inseawater, whilethethick, waxyandresinous epi-dermisrepelswater(Dehganand Yuen,1983).Species ofCycas thatlackaspongytissuedonothavebuoyantseeds(Dehganand Yuen,1983).WesuggestthatthespongytissueofLunariaallows seed buoyancy in this taxon, as the epidermis and/or the pal-isadelayercouldblockwaterfromenteringtheporousregions. Throughoutdevelopmentandevenatmaturity,whentheseedis dry,clustersofdepositsintheintercellularspacesandliningthe cellwallsofthespongytissue areFluorolYellowpositive, indi-catingthattheyarelipids.However,thesediscretedropletstend todisappearwhentheseedundergoeshydration,astheFluorol Yellow stainingbecomesmore diffuseand evidentin thewalls themselves,andthewallsalsobecomediffuselySudanIIIpositive. ThediffusepositivereactiontobothFluorolYellowandSudanIII post-hydrationindicatesthatthelipiddropletsbreakup, releas-ingtheircontents,whichinturnbecomemorereadilyaccessed bythestains.Theselipidsmightprotectthecellsagainst mechan-ical damage due to friction between the walls of the swelling cells.

Pre-sclerenchymatictissue/sclerenchymatictissue

Thecellsofthepre-sclerenchymatictissuehavelarge electron-densemassesintheirvacuoles.Such massescanbeinterpreted as phenolic compounds, probably tannins, as their ultrastruc-tureismorphologicallyverysimilartothat describedfor many plants. Analogousdescriptions have beengiven for the pheno-licsinthecorticalcells ofReaumuriapalestina(Ginzburg,1967), in the endoderm of Gossypium hirsutum and Musa acuminata (MuellerandGreenwood,1978),inmother tannincellsof Sam-bucus racemosa (Zobel, 1985), in the coenocytes of the same species (Zobel, 1986), in the root cap cells of Brassica napus (Zobel, 1989) and in the nectaries of Selenicereus grandiﬂorus, wherephenolsweredetectedwithGC/MSanalysis(Mostietal., 2001).

AsinthecoenocytesofSambucusracemosa(Zobel,1986),the centralvacuolescontainingtheelectron-densemassesinthe pre-sclerenchymatictissue ofLunaria appeartogrowas a resultof fusionwithothersmallervacuoles.Thevesiclesresponsibleforthe primarytanninaccumulationmayderiveinpartfromER cister-nae,assuggestedbyZobel(1986).HoweverinL.annuawenoted thatalsolargedictyosomevesiclescontainingdropletsof electron-densematerialapparentlyfusewiththetonoplastofthevacuoles. Adictyosomeoriginoftannin-containingvacuoleswasfirst pro-posedbyJuhashetal.(1970)inCornus.Zobel(1985)alsoobserved vesiclesleavingtheproximalfaceofdictyosomesinthemother tannincellsofSambucus.Asproposedforrootendodermof Gossyp-iumandMusa(MuellerandGreenwood,1978),theaccumulation oftanninsinthepre-sclerenchymaticlayerofLunariacouldprotect againstpathogens(Krausetal.,2003).Condensedtanninswithin vacuolescouldalsoplayaroleinseeddormancyandgerminationas isthecasewithArabidopsis(Debeaujonetal.,2001).Whenthe pre-sclerenchymatictissueisinthelaterstagesofitsdevelopmentthe cytoplasmbecomesmoreosmiophilic,showingsignsof degenera-tion.Suchmodificationsappearrelatedtothelignificationofsome ofthecytoplasmiccomponents,eventuallyleadingtothe forma-tionofsolidphloroglucinol-positiveblocksatmaturity(asshown inESEMimages).

Conclusions

ThetissuesofthetegumentsofL.annua,bothwhenunripeand atmaturity,possesscomplexmorphologicalaspectsrelated even-tuallytotheplant’sbiologyandecology.Alltissuesofthetestaare deadatmaturity,potentiallycreatingarelativelylighterseed.The spongytissuecouldbeinterpretedasanadaptationoftheseedto increasebuoyancyandfloatationcapabilityfortransportinwater duringtherainyseason.Thefeaturesofthisspongytissueindicate thatLunariaisadaptedtoandthusmayhaveevolvedina Mediter-raneanenvironment,whichischaracterizedbyalargeamountof rainfallconcentratedinshortperiods.Suchconcentratedrainfall reactivatesdriedstreamsalongwhichtheindividualsofL.annua regularlygrow,andwherethetypicalenlargedandflattenedfruits ofLunariacouldeasilyfloatandbedispersedawayfromthemother plant,whiletheseedsbecomeremovedfromthereplumduringthis transport.Thematuresclerenchymaticlayerrepresentsarobust layerofprotectionfortheembryoinadditiontothatprovidedby themoreexternaltegumentallayers.Thesclerenchymaticlayer, beingthickened,is likelyelasticdue toitsarticulatedstructure. Theelasticitywouldallowtheembryotoswellduringhydration withoutbreakingtheprotectivetissue,atleastatthebeginningof itsswellingphase.

Acknowledgements

WethanktheCassadiRisparmiodiPistoiaandPesciaforthe ﬁnancingofthePISTOLIOproject,whichallowedustoinvestigate seeddevelopmentinsomemembersoftheBrassicaceae.

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