Micron and MicroscopicaAda. Vol. 15. No.3,pp. 153-160, 1984. 0739 6260,84S3.00+0.00Printed in Great Britain. 984 PergarnonPressLtd

COMPARISON OF TRACHEARY ELEMENT DIFFERENTIATION ININTACT LEAVES AND ISOLATED MESOPHYLL CELLS OF ZINNIA

ELEGANS

JEREMY BURGESSand PAUL LINSTEAD

Departmentof Cell Biology, John Innes Institute,Colney Lane,Norwich NR4 7UH. U.K.

(Receited24 February 1984)

Abstract—Theprocessof trachearyelementdifferentiationin intact leavesof Zinniaele,qansisdescribedusingtransmissionelectronmicroscopy.Theprocesscanbedivided into threephases:wall thickening,cytoplasmicdegenerationand wall hydrolysis. Thesefeaturesaredescribedandcomparedwith similar stagesoccurringduring the differentiation of trachearyelementsfrom isolated Zinnia mesophyll cells. This comparisonemphasisesthat theunusualfeaturesof differentiationseenin isolatedcellsarenot typicalof thesameplant’sbehaviourduring normalgrowth. Thepossiblenatureof theobserveddifferencesis discussed.

Index key words:Differentiation, cell wall, Zinnia, xylem.

INTRODUCTION appearsto becontributedto thewall spacein the

It hasrecentlybeenshownthatmesophyllcells form of vesiclesproducedby activedictyosomes,isolatedfrom youngleavesof Zinnia elegansare which aboundin thecytoplasmduringthisphaseableto differentiateinto trachearyelementswhen (Hepler and Newcomb, 1964; Pickett-Heaps,culturedunderappropriateconditions(Fukuda 1966). As the thickeningsmaturethey becomeand Komamine, 1980; Burgessand Linstead, lignified, and cytoplasmicdegenerationfollows.1984).This behaviouris exhibitedby morethan Thefinal stateof thematuretrachearyelementishalfthecells in thepopulationandthusprovides reachedfollowing hydrolysisofthosepartsof thea potentially useful systemfor the study of a wall which are not thickenedandlignified. Thedevelopmentalprocessin vitro. Differentiationis cell thusproducedforms part of a continuousboth synchronous and rapid, and this has systemof connectedporoustubes.enableda description of some of its structural Thesethreephasescan also be recognizedinaspects(BurgessandLinstead,1984). liquid culture in vitro. The process of wall

The formation of tracheary elements in hydrolysisseemsto be unusualsinceit proceedsprimary xylem of intact tissueshasbeenoneof through the formation of a particulate hydro-the mostwell studieddifferentiationpathwaysin lysed wall rather than the widely describedhigher plants,and the subjectof many articles fibrillar intermediate stage (O’Brien, 1981).and reviews (e.g. seeO’Brien, 1981).In general Clearly such differencesas exist between thethereis a highdegreeof consensusconcerningits generalschemefor xylogenesisandthe in vitrovarious stages.Theseare convenientlydivided description(BurgessandLinstead,1984) mightinto threephases:wall thickening,cytoplasmic representa result of experimentalmanipulation.degenerationand wall hydrolysis. Localized Alternatively it is possiblethat xylogenesisinformationof wall thickeningsis associatedwith intactZinnia leavesis in somewaydifferentfromcytoplasmic localization of microtubules the generalizeddescription.The presentpaper(CronshawandBouck,1965)andacharacteristic reportsthe courseof the formationof trachearypattern in the distribution of endoplasmicre- elementsin intact leavesof Zinnia,andcomparesticulum (Pickett-Heapsand Northcote, 1966). the processwith the in vitro systemof differenti-Material for the growth of the thickenings ation from mesophyllcells.

153

154 J. BurgessandP. Linstead

MATERIALS AND METHODS non-differentiating cells are usually randomly

SeedsofZinnia elegans,cv Envy (SuttonSeeds, disposedalongthe longitudinalwalls. However,Torquay, U.K.) were germinated on moist as Fig. 1 alsoshows,it is possiblein developingfilter paper for 48 h, then planted into a peat vasculartissueto find cells in which groupingofcompost (Levington’s compost, Fisons Ltd., themicrotubulesis apparentevenin theabsenceIpswich, U.K.) in 9 cm plasticpots,oneseedling of any visible wall thickening. This effect isto a pot. Youngleaves,2-3 cm long from three- consistent with the idea that regrouping ofweek-oldplantswereusedto providespecimens microtubulesfrom their customarynon-orderedfor fixation. Leaf pieceswerefixed in cacodylate positions in non-differentiatingcells may be abuffered 5% glutaraldehyde,pH 7, at room veryearlysign of differentiationwhich precedestemperaturefor 1 h. The fixative was infiltrated secondarywall deposition.It also illustratesthein vacuo.Specimenswerethenwashedin distilled possibilityof cell—cell interactionin theposition-water overnight at 5°C,postfixed in veronal ing of wall thickenings;thesefrequently occurbuffered 1 % osmic acid at 0°Cfor 1 h, and oppositeoneanotherinadjacentcells (Figs.2, 11,dehydratedat 0°Cin a gradedethanolseries. 12).Specimenswere then embeddedin Araldite At the early stageof differentiationshown in(CIBA Geigy, Duxford, U.K.) and sectioned. Fig. 1, very little cytoplasmicspecializationisSectionswereexaminedin a JEOL EM 100B at evident other than the localization of micro-60 kV, afterstainingwith uranylacetate(20 mm) tubules. Short fragments of endoplasmicre-andleadcitrate(2 mm). ticulum (ER) cisternaeare visible close to the

wall, but their positionsdo notseemto correlatewith the developingthickenings.As the thicken-

RESULTS ingprocesscontinueshoweversuchERcisternaeareincreasinglyfoundto lie betweenthethicken-

(a) Wall thickening ings (Fig. 2). Thesourceof thematerialwhich isTheappearanceof smallincipient thickenings incorporatedinto the thickeningwall is notclear

is the first reliablestructuralsign thata cell is to from fixed sections. The appearanceof largebecomea trachearyelement. The position of numbersof cytoplasmicvesiclesdoesnot occurthesesmall thickeningscoincideswith the ap- until quite an advancedstagein the thickeningpearanceof localizedgroupingsof microtubules processis reached(Figs.3, 5). Active dictyosomesin the cytoplasmover the thickeningpart of the are commonly seen in the cytoplasm by thiswall (Fig. 1). This patternof microtubuleswas stage,andthe ERcisternaewhich lie betweenthealwaysobserved,no matterhow smallthe degree thickeningsarefrequentlydistendedandcontainof wall thickening.Microtubulesin non-dividing, granularamorphousmaterial(Fig. 3). Theedges

Legendfor page 155

Fig. 1. Electronmicrographofthewall betweentwo cells in thexylem ofa young Zinnia leaf.Small thickeningsassociatedwith localizedgroupsof microtubulesarevisible in theuppercell. Localizationof microtubuleshas

also takenplacein thelower cell. x 47,500.

Fig. 2. Electronmicrographof thewall betweentwo adjacenttrachearyelementsin thexylemof ayoungZinnialeaf. In theuppercell whichis still developing,microtubulesarelocalizedoverthethickeningregion ofthewall.

Profiles of endoplasmicreticulum arevisible on either side of thethickening. x 38,000.

Fig.3. Electronmicrographofthewall betweentwo cellsin thexylem of a youngZinnialeaf.At this laterstageindevelopmentto thatshownin Fig. 2, theendoplasmicreticulumin thedifferentiatingtrachearyelementmaybe

distendedin sectionandclosely applied to the baseof thewall thickenings. x 19,000.

Fig.4. Electronmicrographof aglancingsectionacrossa developingtrachearyelementin a young Zinnia leaf.Largedistendedprofiles of endoplasmicreticulumcontainingan amorphousgranularmaterial arepositionedbetweenseveralof thethickenings.The membraneof theendoplasmicreticulum is fragmentedat this stage.

x 8550.

Fig.5. Electronmicrographof adevelopingtrachearyelementin thexylem ofayoung Zinnialeaf.The dumb-bellshapedproplastidin thecytoplasmis apparentlyaboutto divide by a fission process.x 19,000.

Zinnia TrachearyElementDifferentiation 155

~~•t ~ ~ ~ ~ •~‘ ~ “~

~5~~ r

-2~

~

L I:~2 3 ~

~ ~

156 J. Burgessand P. Linstead

of thesedistendedcisternaeareoften seenclosely resultof the directionof flow of fixative into theappliedto thebaseof thethickenings(Fig. 3). As cell during the original fixation procedure.It isthe differentiating cell matures the depth of widely heldthat oncemembraneintegritybeginsstainingwithin the wall increases.Thisincrease to be lost in amaturingtrachearyelement,totaldoesnot only occurwithin the thickenings(Fig. degenerationof cytoplasmic structure occurs4) and therefore probably does not represent rapidly thereafter. It is becauseof this thatsolelyan effectof lignin deposition. intermediatestagesare alwaysdifficult to ob-

serve.In Fig. 7 thetonoplasthasdisappearedand(b) Cytoplasmicdegeneration the nucleus,thoughstill clearly identifiable,hasIn the intact leaf, cells which are to undergo lost its long lenticular shapeandis coatedwithdifferentiation to tracheary elementsdo not cytoplasmic remnants. Small cytoplastic or-developlargepopulationsof chloroplasts.These ganellesare still identifiableat this stage,havingorganellescannotthereforebeusedasanindexof cometo rest betweenthe wall thickenings(Fig.the progress of cytoplasmic degenerationas 8). Later stagesthan this in the degenerationofseems possiblewith differentiating mesophyll the cytoplasm have not been observed. It iscells (Burgess and Linstead, 1984). Indeed, it sometimespossibleto seestainedmaterialwhichappearslikely that proplastidscontinueto divide possibly correspondsto the plasma membranein differentiating cells within intact leaves,de- overlying the wall thickeningsduring hydrolysisspitethefactthat totalcytoplasmicdegeneration of the wall (Fig. 11), but generallyspeakingallis imminent (Fig. 5). The nucleusof a maturing membranecomponentsare lost by this time.trachearyelementistypically longandnarrowinsection,with amarkedperipheraldistribution of (c) Wall hydrolysischromatinandanintactnucleolus(Fig. 6). As the In common with other intact systems,wallprocessof wall thickeningiscompleted,it becomes thickening and cytoplasmic degenerationarenoticeablymoredifficult to preservecytoplasmic followed by hydrolysis,of thosepartsof the wallmembranesin an intact state.Thus in Fig. 4 the whichare not requiredfor thefunctioningof themembranessurroundingthe contentsof the ER maturetrachearyelement.In primary xylem inare brokenin many places,andin Fig. 6 leakage leaves, this means that eventually only theof cytoplasmicmaterialsinto the vacuoleseems lignified thickenings are retained. Hydrolysisto havetakenplace.It is occasionallyobserved doesnotappearto beginuntil thecytoplasmhasthat preservationof membranesis not uniform completelydegenerated.Figures 7 and 8 showalong the length of a single cell. For example, that the plasmamembraneremainsvisible aftertonoplastmembranepreservationmay be excel- the lossof mostof thecell contents,andwhilst it islentatoneendof amaturingcell,with no obvious still present,no signsof hydrolysisare observed.boundarybetweenvacuoleandcytoplasmat the This is in contrast to the results of Esau andotherend.This might representa real effect of Charvat (1978). Hydrolysis proceedsfrom thepolarity in the timing of the autolysis of the surfaceof the primarywall betweenthe thicken-cytoplasm,but it is much more likely to be a ings (Fig. 9). Thehydrolysedwall is swollen and

Legendfor page 157

Fig.6. Electronmicrographofa developingtrachearyelementin thexylemof ayoung Zinnialeaf.Thetrachearvelementnucleusis characteristicallylong and narrow in sectionwith ratherpointed ends. x4I80.

Fig. 7. Electronmicrographof adevelopingtrachearyelementin thexylem of a young Zinnialeaf.Cytoplasmicdegenerationhas takenplace,leavingthenucleusless elongatein shapeand coatedwith cytoplasmicdebris.

x 14,250.

Fig. 8. Electronmicrographof aglancingsectionacrossadevelopingtrachearyelementin thexylem of ayoungZinnialeaf.A few organdIespersistin thecell lumen,and theplasmamembraneremainsmoreor lessintact at

this stage. x 14,250.

Fig.9. Electronmicrographofaglancingsectionthroughadevelopingtrachearyelementin thexylem ofayoungZinnia leaf.Thewall on eithersideof the thickenedregionsis swollen andundergoingdispersalinto the cell

lumen. x 15,010.

Zin,iia rrachearyElement Ditierentiation 157

‘.4*9 ~

~ .‘: ‘~ .

C. ~ . .~‘

0

~ ~

11 _

158 J. BurgessandP. Linstead

has a coarsely fibrillar appearance.Fibrillar clearlyprecedeswall developmentasmeasuredbymaterialappearsto breakawayfrom the surface fluorescentdyes (Linstead et al., unpublished).of thewall andenterthelumenof thecell (Fig. 9). Theserelative timings do not contradict theWhen the adjacent cell is not differentiated, widely held view of a functionallink betweenthehydrolysisof the wall on the differentiatedside groupingof microtubulesin thecytoplasmandonly proceedsasfar asthemiddlelamella,which theformationof alocalizedthickeningin thewallasa resultappearsasadarkstainingline (Fig. 9). (Pickett-Heaps,1967;HeplerandFosket,1971).Betweenadjacentcells which haveboth differ- However, equally they do not confirm a causeentiated,the middle lamella may persist for a andeffectrelationshipbetweenthesetwo pheno-time as a visible feature betweenthe basesof mena.Similar considerationsapplyto the roleofpairedthickenings(Fig. 10). Thefibrils compris- the variouscytoplasmicmembranesystemsdur-ingthehydrolysedwall do notresemblecellulose. ing wall thickening.It is certainlycharacteristicThey are well stained,not necessarilystraight, of developingtrachearyelementsthat the cyto-maybe branched,andare thickerthancellulose plasm contains active dictyosomesand manymicrofibrils of higher plants. In the hydrolysed vesicles.However,therelationshipbetweenthesewall they form a loose network which also components,the endoplasmicreticulumandthecontains finely granular amorphousmaterial wall, is far from clear and has not been con-(Fig. 10).This networkeventuallybecomesvery clusivelyelucidatedevenusingradioautographictenuous,and the contrastbetweenthe hydro- techniques(Pickett-Heaps,1966).The degreeoflysed and lignified parts of the wall is marked this activity isgreaterin intact leavesthanit is in(Fig. 11). In the maturetrachearyelementthe isolatedmesophyllcells, despitethefact that thehydrolysedwall is completelyabsent,and only time scalefor full differentiation of a trachearythethickeningsremain.As a comparison,Fig. 12 elementseemsto be similar in both situationsshowspart of the wall of a maturing tracheary (Sachs,1981; Burgessand Linstead,1984).Thiselementwhich hasformedoveran8 day period might betakento imply that thegreateractivityfrom an isolated mesophyll cell of Zinnia (full seenin cells within intact tissuesis not entirelydetailsin BurgessandLinstead,1984).Thewall related to wall developmentalone. O’Brienthickening has retained its integrity at this (1981)lists a varietyof otherfunctionsthat suchadvanced stage of wall hydrolysis, but the cytoplasmiccomponentsmight be performing,unthickenedwall on either side and beneathit and thesemight not necessarilytake place inhas beenchangedto a structureconsistingof isolatedcells.granular material with no apparent fibrillar Degenerationof the cytoplasmtowardsthecomponentat all. end of the differentiation processseemsto be

rapid. It is difficult to assesswhether stagesduringwhich membranecomponentsarepoorly

DISCUSSION preservedrepresent a true reflection of cellTheseresultsconfirmthatxylogenesisin intact physiology.In this regardthe appearanceof the

Zinnia leavesis by and large similar to the ER is of someinterest.Its laterform, asswollengeneralizeddescriptionof theprocessoutlinedin cisternaewhich are oftenclosely appliedto thethe literature(O’Brien, 1981).They suggestthat base of wall thickenings, is not observed inthe unusualpathwayof wall hydrolysisobserved differentiatingmesophyllcells in vitro. In differ-in the developmentof trachearyelementsfrom entiatingvesselselementsin corn,Srivastavaandisolatedmesophyllcells of Zinnia is a responseto Singh (1972) appearto incline to the view thatthe experimentalconditionsused(Burgessand swellingof theER andlossof ribosomesfrom itsLinstead,1984). surfacerepresentdegenerativechanges.In the

Cytoplasmiceventsconcernedwith wall thick- caseof Zinnia, the modification of the ER is soening seemto be comparablein the two situ- strikingandapparentlyorderedthat it isfeasableations. In vivo it is possible to infer that the totaketheview that ratherthanresultingfrom aredistributionof microtubuleswhich is observed degenerativechange,this modification may beduring wall thickeningin fact takesplacebefore relatedto its inception.any thickeningoccurs (cf. Fig. 1). This is more The most striking difference between cellsconvincinglydemonstratedin isolatedmesophyll within intact leavesandisolatedmesophyllcellscells, wherethe distribution of microtubulesas concernsthe hydrolysisof the wall at the endofvisualized by fluorescent antibody techniques trachearyelementdifferentiation.Hydrolysis in

Zinnia TrachearyElementDifferentiation 159

~ ~

~‘ .~~~

I ~..‘

12 ‘~:~‘.

Fig. 10. Electronmicrographofthe wall betweentwo adjacenttrachearyelementsin thexylemof ayoung Zinnialeaf.Theprimarywall betweenthethickeningshasbeentransformedinto aloosefibrillar network.Thedarklinebetweenthebasesof pairedthickeningsrepresentsthemiddlelamella of theoriginal primary wall. x 30,400.

Fig. 11. Electronmicrographof agroupof developingtrachearyelementsin thexylem of a young Zinnia leaf.Thecontrastbetweenthe unhydrolysedlignified partsof thewall and thehydrolysedfibrillar wall is marked.x 7125.

Fig. 12. Electronmierographof thewall surroundingan isolatedZinniamesophyll cell after8 days in culture.Thecell hasdifferentiatedinto atraehearyelement.Theoriginal primarywall hasbeentransformedinto aloose

aggregationof granularparticulatematerial. x 12,350.

160 J. Burgessand P. Linstead

vivo leadsto the formation of a loosely fibrillar know thechemicalnatureof thehydrolysedwallwall which swells into the lumen of the cell and in the differentiating trachearyelementsboth inthereapparentlydisintegrates.This appearance vivo and in vitro. This is particularly interestingof the hydrolysed wall is widely observed(e.g. since the formation of tracheary elementsbyO’Brien and Thimann, 1967). The chemical intact leavesandisolatedcells of Zinnia elegansnatureof the hydrolysedwall is quite uncertain, representsthe attainmentof a particular differ-In the presentcase,although it may be fairly entiatedstateby routeswhich differ in severalofdescribedas ‘fibrillar’, the fibrils which comprise their structuraldetails.it in no way resemblecellulosemicrofibrils. It islikely that the appearanceof thehydrolysedwall REFERENCESresults from the staining both of a fibrillarbranchedcomponent,andalso a finely granular Benayoun. J.. 1983. A cytochemical study of cell ssallhydrolysisin thesecondaryxylem of thepoplar Populu.samorphouscomponent.By contrastthe hydro- italica Moench) Ann Bot., 52: 189200lysed wall formed by in vitro developmentof Burgess, J. and Linstead, P. J., 1984. in i’itra trachearytrachearyelementsfrom mesophyllcells is coar- element formation: Structural studies and the effect ofsely granular and finally lacks any fibrillar triodobenzoicacid. Planta, in press.

Cronshaw,J. and Bouck,G. B., 1965. The fine structureofcomponentsat all. The physicalresult of these differentiatingxylem elementsJ Cell Biol., 24: 415 431apparentlydifferent chemical processesis the Dawson,A. L. andGahan,P.B., 1979 Longevityofhydrolasesame:that the hydrolysedpartsof the wall lose moleculesduringprimary xylem differentiation..4nn. But..their mechanicalintegrityandareeventuallylost. 42: 251 254.Whether this final dissolution is a chemical Esau,K. andCharvat,I., 1978. On vesselmemberdifferenti-ation in the bean (Phaseolu,srulgari.s L.). ,4nn. Bot.. 42:processor representsmerely the physical re- 665 677moval of fragmentsof materialcannotbe as- Fukuda, H. and Komamine,A.. 1980. Establishmentof ancertained.Theappearanceof fibrillar materialin experimentalsystem for the study of trachearyelementthe lumen of developingcells in vivo suggests differentiationfromsinglecellsisolatedfrom themesophyll

of Zinnia elegan.s.Plant Phy.siol., 65: 57 60.somephysicaldisintegrationdoesoccur during Hepler. P K and Fosket, D E. 1971. The role ofwall hydrolysis. The extent of hydrolysis is microtuhules in vessel member differentiation.limited by two physical circumstances.It does Protoplasnia,72: 212 236.

not proceedinto regionsof the wall which are Hepler,P. K. and Newcomb,E. H., 1964. Microtubulesandlignified the thickenings in the case of the fibrils in thecytoplasmof Coleu.s cells undergoingsecon-

dary wall deposition.i. Cell Biol.. 20: 529 533.primary xylem consideredhere. It does not O’Brien, T. P., 1981. The primary xylem. In: Xylem Cell

penetratebeyondthemiddlelamellainsituations Deielopment.Barnett,J. R. (ed.), CastleHouse,London.whereadifferentiatingcell hasasits neighboura O’Brien, T. P.andThimann,K. V., 1967.Observationson the

cell which hasnot yet developed.The natureof fine structureof the coleoptile. Ill. Correlatedlight andelectronmicroscopyof the vasculartissues.Protopla.~ma,this protectiveeffect of themiddle lamella is not 66: 443 478

known. Its actionpossiblyremovesthe needsfor Pickett-Heaps,J. D., 1966. Incorporationof radioactivityspatial control of the releaseor activation of into wheat xylem walls. Planta, 71: 1 14.hydrolytic enzymes.However,Benayoun(1983) Pickett-Heaps,J. D., 1967. The effectsof colchicineon thehas presentedcytochemicalevidence for such ultrastructureofdividingplantcells,xylemwall deposition

and distribution of cytoplasmicmicrotubules.Dei’. Biol.,spatialcontrol in secondaryxylem of poplar. It 15: 206 236is alsoof interestthathydrolyticenzymeshavean Pickett-Heaps,J. D.andNorthcote,D. H.. 1966.Relationshipactivelife which greatlyexceedsthe period over of cellularorgandIesto theformationanddevelopmentofwhich tracheary element differentiation takes the plant cell wall. J. exp. Bot., 17: 2026.

Sachs,T., 1981. Thecontrolofthepatterneddifferentiationofplace,at least in a form detectableby electron vasculartissues As/i’ But Re’, 9: 151 262microscopy(DawsonandGahan,1979). Srivastava,L. M. and Singh. A. P., 1972. Certain aspectsof

Clearly it is now of considerablerelevanceto xylem differentiationin corn. Can. J. Bot., 50: 1795 1804.