or 01 01

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The Developme nt01Xylemwit hin the Tomato andits Influence on the Movement01Water and Calcium

into the Fruit .

BY CDKathiA.Hud ak,B.Sc. [Honours]

Athesissub mitte dtothe SchoolofGrad ua te Stud ies inpart ial lulfillmentof the

requirementsforthedegree of Master

o r

^Science

Depar tmen t ofBiology Memor ial Univers ityofNewfoundlan d

23Febr uarylOgO

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11+1

NaliOnal l ibfa ry ol'C3nada

Biblioll'l«luc natlOl'l..lc .,C""",,

The eutnor hasgranted an Irrevocable non·

exclusive licenceallowingtheNationalUbrary ofCanadatoreprod uce ,loan,disbibule ()(se~

copiesof htsrher thesisby anymeansandIn any formor format.makingthisthesis available toInterested persons.

The authorretains ownershipal tha copyright in hislher thesis . Neitherthe thesis nor 'SUbstantial extracts 'romitmaybeprinted or otherwise reprod uced withouthislherper- mission.

L'euteura accorde une licencelrrbvocable et nonexclusiveperme neotaIaBibfiol heqoo nationale du Camvh d'1reprcdclre,pret e-, distrlbuOf ouvoodoodeecopiesde satM se de quelque menlere01sous quelquetome que cosoilpourmet tredesexemplairesdo cettethese Aladispositiondespcrsonnc s lntereseees.

L'auteurconserveIaPfopnetedudroitd'aulcur quiprol~~samese.NiIathesenl des extrets substanl iels de ccae-cr no doivcnl etrc im~ouautremcntreproduils sansson autorisation.

Canada

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Abstract

Functional associationsofxylemvascularization,calciumtranspor t,and water nux were studied inLy~o)per8;~cnesculentu mMill.-Tiny Tim- . Xylem format ionof thepedicel and fruitwas traced fromtheemergence of thenor BI primordi athrcugb to the development of matur eIruit.Berberinehernl-sulph ete, anapoplastieallymobile dye,wasusedtofellow water movements throughthe xylemsystems ofintactstemsandtrusses.4SCawasused to ident ifyareasor calcium localizat ion wi'.hintheplantandcorrela ted Withtranspira tionrat es measur edforthe selectedplantparts.

Waternow andcalcium distribut ion in theplant werenotuniformandthe patter n wasdependent ontheextentorxylemvascularization.Leaves,sepals, and the smallest immatu reIruit«30 mm3),whichwer e all well supp lied with xylem,shewedthe great est accumulationsor calcium.Fruitin thisclassalso showedtbe highesttranspirationrates.Incont rast,calciumeonecnt retion was lowest in matur eIruit,whichalsohad the lowest proportionof xylem vascularizationand lowesttranspirationrates.

These findingsbearonthe mecha nismof induction or blossom end rot,a phytopatbological condition regarded as a calcium-deficiency disease ortomatoes.

Thesuggestion thatthe deticiency might tirst occur in very small fruita_'~a result or their early, rapidincreasein volume is notsupportedbythepresent dat awhich demonstrat ethatsucb (ruit,dueto high waternuxand transpi ration rate, accumulatelarge amountsorcalcium.Tbe distalport ion or lar gerCruitmay become calciumdeficient because apoplastic water does notreachtheCar blossom endof these fruit.

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iii Acknowledgements

Ithllnk var ious members offi\~ultywhoassistedin the ereen ce of thiswork:

Myeuper vlsor,Dr.RegerLee,mademeeonsiderahernat.views and ideas.Not Hl'l~·thingi...L"itSCNIU.

Dr.JohnOowr('d ir~I .-dhispath or reseer ehrorsf!nulmonth~to indudetr.Ca dbtrih ut ion intomatoes.

Dr. David"arson,mymentor,untan gledIn)'mess orthought!LSanongoing oeeupet icn,and pointed meio theright direction.

Dr.StevenCarredited tbetbesisandhelped me wit hthefinal product.

Dr. WilliamThrcllallshowedencoura gemen tand confidencein my Abilities,an d keptadministr ators atbay.

Dr.AryaBat and Dr.PeterScott provided editorialcomments.

Jacknowledge Dr.CarolPetersonoftheUnivu sityof Waterlooforteaching me techniqu es ofsta iningeedobserving vasculartissue.

Jam thankfultoMr.RoyFickenforbav ing a remarkably highharassmeot thresholdwhenitcame to multiplepbotographs.

Fra nkBoothroyd,John Christilln.and DarylJonelll toleratedm),ravings oftbe lut severalmonths,tficbel.Normorereinfor ced the prac:ticalmeaningofscience, and ChristineCampbelltaughtme SpssGraph ics.Jam gratefulto thestaffof the QueenEliu betb

n

Library for endlessextensionsof periodicalmaterie l,andto thestArfofComputinsServices forgenerations ofthe!lisecplee putclosing time.

Iamthankfultomyfamily for theirmoralsupport endpatience.

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e.

Literature Cited

Appendh: A.Hoagland's Nutrient Solution A.1. Macto n utrients

A.2.Micronuteients Appendix B.

Appendix C.

Appendix D.

AppendixE.

8.

76 77 87 87

8.

87

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U

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List of Figures

Fisure II A.floralprimordia .B.Entireiuflcreseeuce 22 FJsure 2: A.En tire inflor eseeneewithelements reaching sepal tips. 2'

B.Xylemtissue ol Ibepedicel and sepals.

F1sure3: A.Enlargement of trachearyelementsthrough the pedicel. 2.

B.Mature pedicel.

Figure 41Percent 01xy lemcross-sect ionalarea throughthepedicel. 28 F1su re61 A.Xylempattern

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the sepals.B.Longitudinalmedian 31

sectionofped icel and fruit.

Fisurettl A.Med iansecti on 01 buit.B.Traebearyelements01 33 place nta.

Figure 71 Proxim al(AJabscissional (S}anddistal(C)pedicelcross-section s 3.

illustratingdecr eased xylemattheabscissionzone.

Figure 8: A.Cross-sect ion01 sepa lillustratingxylem vessels. 38 B.Cross-sect ionortheproximalend

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theIruit.

Figure 0: Calium(dpnVrela tiveto total truitwater intakeoverthe <3 tourday experimental period.

Figure 10: Cald um{dp m)relativetogrowth overthe tourday 45 exper iment a l period forall fruitatested .

FI~ure11: Cuticular conductance of truit relative totbeir surface 47 area.

Fila" 12: Relativevolumeincreasecompare dtoinitial Iruit volume. S!

Fllu"181 Calcium contentrelat iveto fruit positionalongthe truss. 5<

Fllure 141 lncresse of volume relativ eto(ruitposition alongthe 5.

trues.

Figure16: Relativegrowthof fruitcomparedto their positionalong 59 the truss.

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List of Tables

Ta ble 1: \!('...n~:'('ll.dpm ,...lu....(.r,,1.101I,ar'~. II Table2: ~iztt! :v;.!'f'!l01 hmu,lofruitwith (hl'itIlll"i\nIr:ansl,ir :alj,," !>O

raft ,nlriu m.C'('umub,l ton.and rrla lj\-l'«reo"'lh.

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Chapter 1 Introdu ction

1.1.Flo ra l De velo p m en t

The inlloeoscenreorthe tomat o1l,y r op~rRiroTit8cutt ntumMill.] origina tes as an elongate dvegetativeapex.Theapexisrleuencd.as istypical

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infkucseenc c initiation. and thefirstflowerisIorm od(rom thisIlaucncddome, Theiudivldualparts oftheIlowerareformed loseries,beginningwit h the developmentorsepalsatthe outer mar gin and progressingtotbecenter where carpelsarcform ed.UsuallyIiveor occas ionallysixsepalprimordi adevelop throughmerist em atie act h'ily,Independent

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eachothe r,at theperiph er y

o r

the nnlt ene dapex,andCorma helixaround the apexmargin.Thesepa lsariseina clock-wisedirection, resulting in a circlearoundthe apexdome(Sawhneyand Greyso n1072).Theirgrowthis more rapidonthe irabaxialsidessothatthe tips inclinetowards each othe rasthey elonga teandpartiallyenclose thecavity beneath them. Aller sepaldirte rentiatio n,acircleorindependentmer istema t ic regions developaltern at etothe sepalsand growinto thepetals,ina similar fashiontothese pals.Thesteminal whorlthendevelops oppositetothecalyx and alternatewiththe corolla.Eachprimordium orthe staminat ewborlconsistsor homogen eoustissue, which asthe tissuegrows,becomesdirrerentiatedinto the distinct par tsorant herand rilament.Therour thwhorlor riveor sixprim or d ia

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develops ink! thefused carpels of the gynoeeium. The placentaon the axillary wallof each~arpelenlarges sothat it, with the dl'veloping ovules and jl'lIy-lih mesoearp ,fillstheloeulesof the carpels.Ovul esereattachedby their funiculu s directl y totheplacent al tissue in aseries of rows.The end rcsultof thisprocew is a pentamerousor hexamer ous,bypog eucu s,ecuncmcrpbt c,bisexualIlower (Haywar d1038).

SubsequentIlcwers ariseaslateralbudsfromtheprecedingIlcwcr and developin8similar Cashion,until8helicoid- cymeresults,usuallyof fiveto seven Ilcwera .The firstIlowertogrowhMadevelopment al advantage over the second, and the second overthethird to theend ctthetruss.Ord ina rily, noraorethan twoIlowere or an inflorescenceare opensimulta neously.Becauseorthis progr essivedevelopment,asinglecymemay have small Cruit,Ilowers, and budsat the same time (Cooper 1027).

1.2.Xylem Development

Xylem differentiationwithin the flowerbud beginsat the inner side or a trace ptocambium near the baseof the bud.It adva nces acropetallytowardsthe bud tipand baeipetellytbrougb the bud basetoconnect to theexistin gxylemor thevegeta tivestem. The patt ernissimilar to thatof a vegetativebud{Jacobs and MorrowHJ57). Severalverticalfilesof tracbeary clements may heinitiated at theisolated locus before on"exte n dsto connectwith the xylemotthe vegetative ste m. Theupwarddifferentiating tilemaynotdirectly connedwith the downwarddifterentiating tile.The two st randsof xylemare then joinedlat er ally bythe ditterentiati on oithe Iew inte rvening procam bial cells to torma short chain of connecting xylemcells (Jacob! and MorrowIg&7).

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Therir"tx)'I"/lltbe.neto Ior rn·'I.tthebase(,fthenOWNbudISthe I,r,,'uxy!l·m.whichmat uresbeforethenOWNbudhselongate d./\~the cells :vlj:ll'f"nlIht'rrf,lmylt"1n,·"It"nd duringbudt"1" np;al ion,thenon-li,·ingtr:l.('hear y

,lirrNI'fllialf"!'tlllrinll;IHI,If·lo..niPtillnandmat un -safl trlhi..pr()('t~sisfini,.;hl'<l.

Thf·U·rnrt".theeh-ments.,fIhrmetaxylcrn are notdl'!'lro~·etlb~·..tretehingand l'l'r"islinthema tureinOor('S('en('t'.Intilt'eventoflargt'see ondarrgrowth.the mr-tnxylem lI!ill:1l1>'becomesncn-Iunetional.However,inmostflower bUrllland pNlk el!i,itremains11..'1the wit'water-conductingtissue(E!illuIgSJI.

The protoxylcm usua lly containsrelativel yfewtrachcaryelements,anda I:ugepro por tionofperenehyma celts,Themetaxylem isa more complextissue whosetraehearyelementsareg4.'n4.'rallywider andart' eeeornpeniedbyfibres along with pu('n('hym.lcells.The highprcpor ticeofcellswithsecondary eell wall,gives thf:mctaxylt'mamore compactandsturdy appearancethanthe protoxylem (Euu 1053).

Oeeenor . l developmentiscomplete,• eontin ucuecylinde rof metuylem occursthroughthelengt hofthe pedicel, whi('hisformed bythebundles ofvesseb and tracheid sandeellsof fibres and xylem parenchyma.Itea elcsesthe interna l phloem and pit h andi,borderedby the extern alphloem ,allofwhiehis sur roundedbycor tex.Thisxylemreachestheeecepts eleorpointofattachment tothe noral parts .Trace,diver ge into thesepals,perels, ant heu, and gyncecium.

Each sep alusu allyhtwlasmaeytracesasalufofthe'a rneplant.Tract'!then breechinto theeorclte,fro mthemetaxylemcylinder,u, uaUyonetoeachpet .t.

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Al'inr;lt' xylt'mtracetr.avelsthrlengthoftill'starnen. Severalx~'I\'11Il,un lllt",m,,~' fr edint o theClHllt'I~:lhr l)"piralnumberisthreelrar~10 eaehrarpl"wilhsmnll b-anehosronnr rlinll:IhrrlHpt'lIary'syste m 10theevuh-s.Thr-rr-arr-:I1~l'tr:mds ofs yk-mIr a d in!!:InIhf'~lil; lll:l.IElIau11:(;')3 ).

traeheids.fibres.andparrnrh>'ma('rll~.Thelr3('hr :lfY't'!l'nu'nb(n'ssl'!sand trach eids] are the watcr-eonduetlng portions of Ihillli"'~lJ('.\'rs.~t·lsc(lnsbl Ilf series of individualcells,thevesselelement s,whoseend walls are partl y(If

completely dissolvedatlaterstnges ofc('11rnnturntion ,1I1l1~Iormlngtilt'lo ng Opt'n vesse ls(seeZimmerma nn108.1).Thetracheiti s arc uSllallymuchlongl'rthan vessel elements andconnectacrosscellwalls withborderedpits.Bothtyp t'll nf cells hav e seconda rywallthickenin gs that ena blethrm toretaintheirshapewhr-n dead,despite the pressureor the surround ingcells(Aloni)gS7).

1.3.WaterTra nsp ort into end thr ough the Xylem

Therate anddirectionof waterflowthrough vesselelements andtraeheids

isdepende nton water pote ntia l Waterpotentialisexp ressed as thecombi ned

effectsofosmotic,turg or,and matrix poten ti3ls.Osmoti c polentialilla funct ion of soluteconcent rat ionand ismeasuredag:lin slareference potentlalofpure waterat atmosphericpressure.This referenceist&~entoherero,thereforethe osmot icpote ntia lofanysolutioni.~alwaysnegativ e. The moreconcentraleda solutio n,tbeloweritsosmotic potentiel. Pressurepot enti alistheresult ofturgor pressureswithincells,balancingthe differencebetween the osmotic andtotal wat er potential,and transpirati onalpullswitb invu culllrchannels.Thematrix

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I'0tl!llliolloradhesion or waterto SUrraCI'Ssuch as c('11 wallsis typicnlly n('gligibll;' inside the plantthat is notin3waterstressed condition. Hence,water movement is dir('ctional and isdefinedbythe magnitude orthegradientof waterpotr-ntink wa\f'fIlIW Il)'Smovestowardthoregionoflowestpotentia l[see BOYl'rIg~.'il.

Till'fMcofwaternux acrosstherootorthe plantand in thexylem Irnchcnryelements ofthesternis determinedbytheroot pressureduringperiods ofdarknessandlowwater stress and therate of transpirationfrom thesurfaceof the plantduring the day (seeMarschner1086).

Soilsolution,which containsdissolvedions,diffusesinto the cortica lcell regionandtravelsepoplesticallythrough thecellwalls ofthecortex.Symplastic movement through plas modesmatainto endodermalcellsmustalsooccur,as water passagethroughthe apoplastis blockedby the suberin-impregnated Cesparten bandsurroun dingtheendoderma lcells(see Clarkson 1084,Peterson 1088).Onceacross the barr ier , ionsare released againinto theapoplastof the stele.Secondaryand tertiarywall buildingof the eadodermalcells01mature root! may destroytheattachme ntoftheplasmalemma and the Casparianband, openingsmallapoplasticchanne lsinthe planeofthe endodermalcellsto allow io n passage(Sanderson1083).Thill nux of ions intothe stele resultsin loweredion concentr a tionofthecortexrelat ive to thatofthesteleandestab lishesan osmotic gradient across this regionJromcortexto stele. Thewat erpotential of the cortex isthen higher than in the steleandcauses watertodiffuseinto it.This diffusion ill!lurricicntto establisha substan tial hydr ostaticpressure in thestele,resultingin water nowup tbe xylemelements01the ste m(BarnlQ66). Thisgradientresults

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in thephenomen on of footIlr('S~urewhich RI,)IH'isinsuHicit'lltto rnusvIlw movement of water thro ughthl.'plant ...it hout thegfl'Rtt'T('!fl'rlsoftfn n~l'irat iol1 trorntbeplant surface.

Trrm- pim t lon crea tesanegativepr rl'.~ ll t ('with intilt'\'.':-'~I'II' lt'll1t'll l,;\n,1 trachcidsdueto cvnpo ratlon Irom!t'lln 's an"('1l\1~t'~water I"11l'J I1l11.',1In-m tilt' lowerstern10theevapora tingsurfacessuchMl<'fll'{'Sandsepals.111'11('<',lin increase in the transpirationrateenhances both theuptakeand the trlll\slo(,l\lion ofmineral elementsin thexylem(seeMarschner(QS6).Therate of lranspiution ISdepend ent onsevl'!ra l factors, suchaslime ofday,relativehumidity, age of tile ph'Il~,andsolut econcentr ation ofthe absorb ed soil solution. Lean s crt-ate the greatest transpirationa lpullandmore thanYO%ofthis transpirationissto m atal.

Tran spirati onratesand movement ofminer alelements are much higher during the daythanduringthe dark period,due tothe opening ofstomataforg:L'!

exch angein photosynthe sisand energyavailabilityfor the waterphasechange.

Short termdeclinesinthetranslocationrates ofminerals at the onset of darkness reflect the changefrom transpira tiontoroo t pressure-mediatedvolume now in the xylem[CrosserIOB8).Anincreaseinthe relativehumidityof theenvironment willdecreasetranspi rationratesfrom the plantsurface as thewater potential difference betw een theplanttissueand theatmosphere decreases. Similarly , an increasein soluteconcent ra tion ofthesoilsolutiondecreasesits osmot ic potential.

However,soil solutionsarerarelysoconcentrat edthat they inh ibit transpiration from tbe leaves (see Boyer}OS5).In seedlings andveryyoung plants ,the effects oftranspirationare small becauseleaf surfaceareaissmall:water uptakeand

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transportto the shootsis determinedprimarilyby thetoo t pressure[see MnrsehnerHI86).

1.4 .Tra ns p ort

or

Ca lci u m

Unlike themajority ormineralelements,calcium istranslocatedinthe xyh-mrnthorthan thephloem.Theconcentra tions(ifallsolutesexceptcalcium nrc severaltimes greate rinthephloemexudate thanin thexylemexudate.The rate ofuptakeandtrans portin the cytoplasmistherefor e severely restri ct edand itis the onlymineral nut rient ot her thanboronthat exists mainly outside the cytoplasmin theapcplas t , Calcium tran sporthas been examinedinstemsof Nieotia'l a L.[HockinglOBO) andin thepeduncles oflup ins,Lupin u8albuIIL., (Pateetaf.1074) andcalciumisconsidereda xylem-mobileminera l with trace amounts,ifany,foundinthephloem.Redlctrecer studies have sho wn that eelclumdocs move intophloem tissue but itisDottranslocated(Biddulphet al.

10S0jandthus only negligibleamounts are everpresent in phloem exudates.

Calcium phosphateisprecipitat edinside the sieveelements inthepresence of high phosphat elevelsand ahigh phloem sappHoC7.5to8.5.Most sieveelements havea filament ousproteinaceous content referredtoas8.p-prot ein ,the appearan ce of which coincideswith the onsetof transloeationwitbinthephloem.

Ithasbeen suggested that contr action andoscillation of thesefilamentspropelthe Iluid or the sievetubes (MaeRobbi et971).Thep-proteina have beenshownto be sensitiveto calciumsimilartoactin-like structural protein s(Kleinig dal.1(71).

Actinisknownto besensitivetocalciumconcent rations (Williamson1075)which may beanoth erreasonCorcalciumexclusion from sieveelements.

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Calcium islIu'r d ofe limitedto tmnslocntioninthexylemandusmtl\'{'nwnt is afunction ofhothm&.~~nowof weter andion exchnngercnctions on the willis oflrllrh('afr elements. 81'11 and Biddulp h(10M ) post ulatedthnteale iumions on entry tothe xylem were adsorbedontonl'gall\'I'I~"rh :'lfll;l·\1~ilt'Shn i n~tll(' illlll'f wallsof theH'SSI'lS. Upwardmovementthrough rhoH._Sl'Iwould occurIL~a transfe r01calcium Irom oneexchange site 10theother.Therute(Ifcalcium ascentwould dependonthe degree oftrnnspirational tensioninsidetheV('s.'l(·ls.

Increasin g trans ptrattcna tpullwould resul tintester w!Ill'r nowthrou gh the xylem andalaster translocation ofcalcium,presumably b('('nusethe ions would ha ve tra velled fartherbeforebei ng readso rbed atanot her('xchangesite (E lIull('rtI06fl).

The!mob ility ofcalciumis alsopro mote dbyothe rdivalentearlo ns that compe te withcalcium for adsorptio nontheexchange sites. Thehigh er the concent rntio n of these cations,the faster themovementor calciumthrough the xylem[Milliknn and HangerIQBB),Also,neu tr ali zat ion ofthecalciumioniccha rge by chelat ion withmalic or citricacids allo wsIreermov emen tofcalcium through the pla nt (Millikan and Hanger HI65 ),

1.5.Function of Calcium

Calciumisa relat ivel ylarg edivalent cationwit hahydratedion icrad ius of 0.412nm.It readilyenters theapopl ast andisbound in anexchangeab le formto cellwallslind the exteriorsurface oftheplasmamem brane(seeMnrs chllerIQsa l.

Moot or its activit yisrelatedtoprov idingstab le butreversibl einter molecular linkag es,predominantlyinthe cell wallsandattbe plasma membra ne. Thus, a high proporti on oftota l cal ciuminplanttissueislocat ed in the cell wallsin

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('f)ntra.~ttoother mncronutricnts .Inthemiddle lamella, itisboundtocarboxylic groups ofpoln.:lIacluronicarid[pectin]whereit ma)'conlain upto50%ofthe tnl:alplanteeleium(Arm~trnn KlindKirkby19 70).Inbot hthemiddlelamella and

"l!\Smamembrane,ealeiumregulat ~membranepermt'a bilit)"andstren gthenseell Willis.InI('a\"('$r('('('ivingahighlevelofeelcium during growth , a !3.rge proportion ofpeeuematerial exists as calciumpeete te.'Ibismakesthetissuefirm and highlyresist anttodl'grad ationby polygalacturonas e(Cassellsand80ulass 1976).Theproportionofcalcium pectatein the cellwalls isalso of importan ce for the ripen ingoffrUits.Rigneyand Wills(I9SI)showedthatduringtomato frui t developm ent,the calci u m con tent of the cellwalls increases uptothefully·

growngreenstage, andsubsequentlydrop s justbeforetheonset ofripeningand sortl'!ningofthetissue.Sim ul taneo usly,ashift in the bindin gst age ofcalcium cceursinwhichwater-solu blecalciumisrever edoverwell-bo un dcalcium.

Com parisonsbetweennormally-ripeningtomatoes andthenonripeningrin mu tan tshowstheim por tance ofcalciumforfruitIirmaess anditssolubilization for fruitripening.rinmutantssbowanincreaseofboundcalcium duringfruit maturation, whereasinothercultivarstbeeceteetofboundcalciumdeclines.

Thisdeclineisassociatedwithan increasein polygalacturonas eacti vit y(Poovaiab 191 0).

Calcium ste biluee cellmembran esbybrid~ngphosphateand carb oxylat e grou ps ofphcephollpldeandproteins at membranesurfaces(Leggedat.198 2).

Asa divalentcation,itreacts with negatively charg edI"hosph.legroupsofthe phos pholipidsinmembranes andstabili zes them.Calci umen ab les membran e!to

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10

Iunct ion lISbarriersag:lill"~uncontrolledpermentionprocesses. Selecti ve ion uptakeattheplasm nlemmnismediatedbycalcium [EpsteinHIIlI).Till' Iu nd nment al toll' of calciuminmembrane st nhilitywnsdemonsmtedhyvnn Com(lfJG~).11"11"Inducedun inerensedll'ak!tg<,of low-molecular-weightsillu lt·~ . mainly potas siumion s,homcellsof calcium-deficient tomatofruits.Pot:\.~si\llll io ns are antagonisticto the functionofenlciu m bf'r(l.us{' of thcir I'0tt' nti:l lin repl ace calciu mon it sbind ingsiteifeaki umconcen trations arc low,whleh...011111 increase cellpermeability(sec BangerthHl79).Increasedrespi rationrate ofthe calcium-deficient tissuealsoresultsfrom10...calcium asIIcons-qeenccof ]eakagr of respiratorysubstratesfromvacuoles\0the respiratory enz y mes inthe cytoplasm[IlangertheIal.ID72j.Thisleakage due tocalciu m drficicncyis similarto the characteristics of tissuesenesce nce. CutcarnationnOWl'n expe rience a 70%declinein ATP-dependentuptakeofcalcium intomieroscmnl vesicles dur ingpost-harvest development Paliyathand Thompson(IQ881suggClll tha t theinhibition of ATP-dependen tcalciumuptake into veslele could be the result oflipidmembranecha nge!that wouldallow calciumion leaka geinto t.he cytoplasmand the rebyfacilitate senescence.

A particu lardisease conditionoftomato,blossomend rot (BER),illustrates the importanceofcalciummovement and the consequence ofits deficiencyinthe pla nt.~the name implies, the conditionischaracterizedbytheappearance of extensive, brownish -blackles ionson the distal endof the developing fruit. Sp urr (1050)described the cellsofaffectedfruits as appeari ngtobeineanadvance d sta teofdisorganizatio n":cells ofthenecroti c tissue collapse, thecytoplasm

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II

coa g ulates, and the nuclei areof abnormal shape. VanGOOf(1068) suggestedthat BEnisIIsymptomofloeelcalciumdeficiency,whichwouldincrease ion pl'rml'ahililyofcellmembra nes,and could aeeountfor tissuenecrosis. BERis ind ucedunderrendi tionsthatarfectthemovement ofcalciumand/oTwaterto thefruit (A rmslrongand Kirkby 107 0,Shayk ewich ellll.10i l,Ward 1073, Wi('fsum1U66).Forrumple,applicationof nitrogen Iertihaerrna)' ormaynot

(':\115('

mm,

dependingon thechemica lformused.Applica tion ofam monium

duringfruit ing induces BER(Taylor and Sm ith 1057) , because

or

in duce d resistanceto waternuxthatreduces thecalci um content oftbefruit (Pilletat.

107 8). Conversely,applicationofnHf.ateresul ts in a highe rorganicacid content ofthe plant,Chela tionof calcium ions by theseacids increasesbothmobilityand concentrati on

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calcium inthe xylemsap (secHa.ng er1070).

Environ mentaleffectsand physiologicalprocessesalsoaffeddistr ibuti on anddeficiencyofcelelum in the tomato.Gerardand Hipp(1068)rep ortedthat an increase in relati vehumid ityreducedtheincidenc e of BER intomatoes,and tha t areductionin lear transpirationenhance dcalciu m movement ink!thefruit.

Brad fieldand Gut b idge(1084 ) foun d thatcalciuminta keinto theto matofruit wasgreaterwhen nightswe r e humidratherthandry endnutrient solutiondilute ra t her thanconcen t ra ted.Positive rootpres sure at night apparently promot es tr a nsportofcalciuminto tiss uesan dorgansthat haverestri cte dtra n spirat ion .

ThisstudyWASmotivatedbythe obser vationthattheextent of xylem eoneentratio nvarie d within andamo ngpla n torgans. Inpnrt icular, xylemis red ucedin the ebseissioezoneof thepedicel . Becausecalciu m,an essenti al plant

(23)

I~

minornl.tanD£'suppliedtovar-iousvla nt pnrts onl,-throughtho apul'la...t,tIll' hnothf'$isofapotentia l restrjctjontoapol'] ~ti('water no\\'eouldbetesfrdhy tr:'ldnl!';thl"developmen t. ofxyll'mwithin thl"flOWNlind fruit,ohsrninv;wntcr flowpntto ms through inl:\\"tplantsegm entsnnd£,,,rtl'latil1F; thiswiththv:'Ifl ':'IS"r

form of lJER.PreviousworksuggeststhatmuchofIhl' W:'Ih'Tsupplil'dtothe fruit arr ivos,'ia the phloem,l's lwl"lal]y during thepnrty

ra...

tgro w th plm,'iI' (Wolterbccketai, lQS7,110et al. IllS;).Presumably,deficiencyofenleium shouldarise atthistime.Thefollowingstud)' attemptstororr e!all"the<':drnt10 whichxylempatt ernand transpirationrail'Sinfluence cnlclumcoueenrea tlon, specificallywithin huit rnnglnginagehomlimeof polfinatjontomaturity,

(24)

13

Chapter 2 Mat erials and M ethods

Tomatoplants,Lyropcrlli conneulenlurnMill.-TinyTim-were gro...n ina 3:1mixtureofpeat basedpottingsoil endvermiculite atp1l6.The plantswere maintain edAtanll\'t'fllgeor65~relat iv e humidityundernuor escenllamps (Syh-nnja,lncnn dcseontfluoresc ent 30W,80/IEs·1rn-:!)with daily16h light 8h dark per iods.TheywerewatereddailyIL!lrequiredandlertiliaed withHoaglan d's solation(Appt'ndi xAlonceeveryrourda)'s.

2.1.Es tablish ment or Xyle mPat tern

Floralprimordiaandinnorescences ofvarioWiages wereexcisedIrom lolIl&toplanta.Tbesewereboileding5% elbanolLoremovepbot05ynthetk pigments,reby dr.W,then cle aredby autoda vinl!:(15mie.liqu idcycle )in10%

KOH.LipiDandcell wanmate rial werestained byautodaving(15 min.liquid

~ycleJina1:1:1solutio no(gly e erol,85%la~liead d,and0.1%c:hlorazolblackE (AlliedChtmica lCo.New York,N.Y.,CJ.No.30235,Lot140 ).Thispreparat ion is amodifieerionof Brun dretlelal.(IQS4).Pedicelewer elongit udinall ybisected todiscern the xylempattern an dwholerruits wereslicedlongit ud inallyinto3mm thiek disb .Plowerbud . werestainedwithout dissecti on.Theplantparts were mounted ina 1:1rat io orglyc:erolandwateran dobservedwit hlight mieroeecpy.

(25)

1·1

~lorphom('l rk An:'l.lpi~W:\.~usedtoestimate);~It'm~IUfllrt·lU I' :\'rd ati n 'I"totnl su rreee.1('&(Tolh1082).MI':L~ur t'ml'nl ~WPI I'1111 111'{rumphO IOltll)l hsufl'1" I'l.f l'11

and stainedf:L.m('nlionn"dl !<I'1l:!.1!'and fruitdi.~k".

noted.Flo" ,""ill'\\a.-t,,.t illl;lh -dbya1I1t":l-" Ufl'lIll' nlIIrIt'lJi:1hInunI',..li.·,·\

altarhmC'nlpointtothe"l'palt ip"',and eonvbined"'ith:l.,li:ulIl'l",1IU':L_ut"lIU' 1I1"I thenoweraC10Mthe middleofthe ovary.Mr :\.'uu'lIll'nbwere takr-nhum enlargedphotographsofIlowcr budsandfruit.Thepatternand distrilJllliollof xylem werestudiedhomtheprimordialstagethroughtothedrnlop m t'nlof maturefruit.The ptcsen ee or absen ceof a pedicelahsdssionzoneW:L.('llrn ·l:tlt>t1 wit hthesizeoftheIlcwer.Lealabscissionzonesw("re :liso elearedand~tll.ilwdin theabove mannerloruse3.~a comparisonwithpcdieelabM'ii'....ionwnt'S.

St rurt ural difCerenr C'S01abscissionzones01the leafAndthefruitwould~ugg(>St possiblelund ionaldirrereoce!between thetwo.Ves-'Ielmemberlength~and diameters within tbeabscissioneon eand theproximal anddulal port ionsof the pedicel weremeasuredfromphotographsoftbetissue.

Pedieelslor estimatio nof tbexylemtiu uecross-~tionalarea...ere harvestedon a weeklybasishom two daysfollowinganthl!9istomat urityateight weeks.Asinglepedicelwassectionedroreeehweek.Tissuesweredehydra tedin an ethanol-butan olseries and embe ddedinparaffinwaxlorseelioni n g(Jensen 1962).

Seria lmicrotomecross-sectionsof40I'm tbicknesswenmadefromth e

(26)

i

s

proxima l to the distal endofeach pedicel. The;ewerestainedwithphlorogtueinel nlHIIICI ,mountedin1%CMC IO,(DOHChemicalsLtd.Poole,EnglRnd)II.non- f(~intJlIs:VpIC'O IlSmountantwitharcfracti ve index of1.3C, prior to I'lifl'''TIlic'rosw p)· .The photographed sectionswr-re projectedontoa digitizi ng appnrntuslind cross-sectionalareasortota lpedicelandxylemwere' measuredin

2.2.Dil"ltributionorFluorescentTracer

Thealkaloidberberi neis anapoplast ical1ymobile dye(SlTuggcr1938)that dot's no l nowthro ughthesymplast ir the tissues are lell intact.The dyeuse d in theseexpe rimen tswasberb erine hemi-sul phata(Sigma,No.B-3376,Lot JiiF·025QI which fluorescesbrightyellow underW epifl uoreeeence. Thedyeis carriedalong the transpir ation streaman d wasusedto show theproportion or xylemthatfunctioned aswater-conducti n g tissue(Dixonand Pet erson19SQ).

Plantstem portionswith attachedtrusseswerecut and theirends imm ersed into0.03%berberineberni-sulpbatein 0.05M pbosphate bullerat pH 6.They werethen leftunderlamps(Sylvania ,Inca ndescen t fluorescent3OW, 80pE s·1 m·2)in an enviro nmentof 20.5 00 and 65%relativehumidityfor3h respectively.

Diamet er or the IruttW8.Smeasur edandCruitcolorW/l!Inotedbeforeimmersi on of stemsindyetojudgeits ripeness and devel opmental stage. Hand erose-sections weremade ortheproximal,middle,and distalportionor thefruitand immediatelyphotogr aphedtoobserve the positionofthe dyeandextentoC lmpregu etlcenJong theCr uitxylem vsseuleture.Photographawerealsotaken or thepedic elandsepal handsectio ns. Thepedunclesof fruit withtheir sepa ls

(27)

lti

thetra nspi rat ionstreamanddyepathway. Thecut ends remainingafh' rM'll:l]

excisionwere eonted wit hpetroleum ;1·11;.: topH'n-nlwaterlossthroughth (,~I' ends. A fluoroseen re standa rd was mad"usmg nh:lI'IIl11I'}"\ OIllI'11'rwithad~' (' concentr-ation(If0.0:11';'andsol ut ion depth(If0.1mill.TIlt,llll':\.~url'd^nU^l.rpsrI'lLt' \' at 6.3x was 20.6.All relativeIluorescencovalues were measuredfitth l' S:U\lI' magnificationanddyeconcent ratio n asth o stan dard.Obst'Hatiun'lwvn-malh' withaZeissPho toscopeIIIusinga Zeissfiller ·187718wit hmaximu mtrans mission betweenanS··&25lim.Thephotoscopewas equippedwith UV t'piOunfl'Sf('lu'l'and an attachedZeissPMl photom et erheadfor thequant ita t ive rluoreseeecc compar-isons.Pluorcscc neevaluesof thestained,wetmountedtissueate represented as"relativeIluor csceneeunits ".

2.3.Distribution

or

45Ca

To mato pla ntstemswit h intactInflorescen ces and leaves wereexcisedlind the ste mends immersedintoHoa gland 's Nutrien tSolut ion cont aini ng0.5 9MJlqor 45Caper 50mlsor nutrj ent solution.Therorm of eelelumusedwas CaCI2lind referen cestocalciumaretotheion.Theseplan t portionswere maintai ned for fourda ys at 21°Cand 65%relat ivehumidity under rlucr eseentlemp e(Sylvllnill., IncandescentIluorescent3OW, 80pE5.1roo'llwithadiurnal16h light An d8h darkperiod. Leaves,petioles,pedieels, sepals,rru iu,flowers, and flowerbuds werethen exc isedand weighed separat e ly. Thefruitwascut intoproxim al and distalhalvesandeach weighed .Fruit wit h a freshweightof lessthnn 0.0 1 gwer e Icrtwhole.Thesefruit ererefe rr edtoas"whol e" infurth ercompar ison s . The

(28)

Il''' ! Ilsed!ur organ eompeeiso noneachstemwasIlmature.full y,up:J.ndl'dIt'a!

do.~l~ t10the fruitin g truss.In eecheasethe petioleWIISthat oftheleafusedin theanalysis .flm....Nsand Ilower budswereIdtintactandincludedsl' p/l I~,pet als, Il.nllll·rs ,andfJ\'ary.Eachpor tion w3.5oven-d ried at60

or

for ·1·.)dr,}"s untilno ehnnge in wr-ightcou ld01'measured.Thedriod sam pleswere thenashedat 650

°c!or:16 h.All.acidextractof the B!3h in50%lIelwasthenoren-dried at60aC.

The remain ing residue was redissolvedin200,.1ofwatcr.Ten m15 ofliquid scintillatio nfluidwas added tothissolutiona.nd thenscintillationcounted between 200and 750kev.Plant portionspreparedintheabove mannerbut without4SC ll.labt'lin g wereusedas con trol sa mples. Scintilla t ioncounts were convertedtodisintegrat ionsperminutea.ndanalysis was based onthes evalues.

Conversions homdpm toemclescalciumwereasfoll ows:

_ Idpm

=

60 dps _I dpa=1 Bq _1480 Bq=1pgCa _ 1 ,.gCaj40.08

=

1 smolCe

Theresultin g proolCa represents the amountatlabeled calcium addedto thenutr ien tsoluti on.The nutr ient solutio n contained12S«molunlabeled calcium.The totnl amountofcalcium in thebeginn ingof the experim ent repr esent edthesumof the labeledandunla b eled cal cium, Calcium was assumed tohe absorbedfromthe solutionat aconst an t rat io of labeledtounlabeled,80 the tots!amo u n tofealeium ineacb plantpartisthe productof smollabeledCa timestheratiootlabeledtctotal calciumat tbebeginningattheexp eriment.

(29)

18

Two measurementsofthelargest fruitdiamet erwere made at right 8ngh's andthe volume of eaehfruit was est imate don the assumptionthatthefruit.is C'Henlill.llyIIsph erelEhrt't sod110HI86a).Volumeswererst im,'lll' \lbelen-and after thefour day experimentalperiodand till' incrrnsosof I-Illumt·""Nt' calculatedasth e dirrercnccsbet weentheir volume sdunngthistune.Hclativo increase ofvolu meis the dillerencein volumeoverthe four0:1)' perioddivided hy theoriginal volume.

frui t or variousages wereharvested,weighed,and theirdiameters measured . Theyweremaintainedat 20°C and65%relativehumidity rcran hour andweig hedat the endofthattime.The differen ce in weight was equaltothe transpir a t ion rateoreecb buit.Water in takeby theIruitisequnlto growt hof therrui t plustheamountofwate r transpired. To estimatetotalwaterint ak e,the unitsoCgrowthmeasuredoverfour days,andtheunits of transpirationmeas ured in hourswere standardizedbymult iplying transpirat ionper hourby Q6.The amountoCtrans p irationinQ6 hou rsbyIICr uitofaparticularvolu meWItSadded to the amo untorgrowtb inIcurdays of a CruitoCtbe samevolume.The epidermalsurfaceofthefruit,sepals,andleaves was exami ned forthe presence of stomat a.

Ananalysisof varia nceorthecalciu mcontentor theplantpartgroup s ...1'..1 usedtoconfirmthehypoth esisofunequalmeans amongleaves, sepals andCruit of diffl!rcntages.Significantdifferen ces in tbe calciumconte ntortbe remaining plant par t group swere shownwith aNew rnan-Ke ulsmultip lerange test.Linear regressionillustratedtherelation ship s amo ngcalciu mconte nt,rel ati vegrowth,

(30)

"

transpira tio n,andfruitvolume.Testswer e doneusingSpssXand Minit ab and graphswere drawn...ithSpssGraphks.

(31)

eo

Chapt er 3 Result s

3.1. EstebllahmeneorXylemPattern

Floral buds of variou sages were examined10follow thestnges of xylem development.Inflorescences, at anearlysinge ofdevelopmen t(wit hntoh.lll'Ili!i1h of 0.4 mm),showedno traceofxylemvesselsbutthe younglonves(Inun in length) surround ingthelntlcresecneee weresuppliedbr cont inuousandconnected sh ands orxylem(FigureI,

AI.

Theelementsdeveloped first in the proximal portionofthe pediceland advanced assinglestra ndsacro pt'tally towardthe"l'pAI tips of tbebud (Figure I,B).Tr acheary elementsappearedinsepalsofbudsthll.t were0.310mlongand were oftenpresentsingly,some were acn-con tinucus (Figure2,

AI.

Continuous strands of vl'S5elsrunn ingthrough the pedieel, sepa" , and petalsandwhich wereconnectedtotbemainnoralaxi" werefirst seenin buds0.610m lo ng (Figure2,B).Theabscissionzonewa.sevidentinbuds 0.8 mm long.The vesselelementsthatfir, tdevelopedwit hin thepedicelwereshort relativetoothervesselclementsof matur e vegetat ivetissueAnd weretwist ed or crooked in shape(Figure3,A). Atbudmalurity,longervesselmembers, tracbeids , And fibresdevelopedalongsidethe croo kedvesselmembers.Atnewer maturity and duringfruit development,onlytwoareasremained composedMllely ofshort,erooked trachear y eleme nts:the pedicelabscissionzoneand thexylem

(32)

21

of thefruitecreIl". dingtotheovules.Stubbyvesselmembersinthe pedicel,

"orderedon thepro xima land distal sidesb)"elongate dvessel mem ber s,well'used 10illi'nl ity thepedieelab~isstonroue.The mean I('nglbof these stubby "eMel members at th .. nbsci<..<;jon zonewa.~0.020 rom ...it harangetromOJXK! romto

0.01;mmcompared withtheves selmembersoftheproximllland distal poriions thailI.vcragedOAmm.The meanwidth of thestubbyvesselmemberswas0.012 romand did not dirterin width homthevessel members proximaland distalto theabscission zone.Theshortvesselmembers werepresentIorthe duration01 frllit development,forming an irregular ring

o r

stra ndsseparatedbypar encbyma cells.Hence,theabscissionzonehadless xylem relat ive tothepor tions ofthe pedicelproximaland dist alto it (Figure3,B).Thisreduction in xyle matthe abscission zoneWASaconsiste nt feature ofallpedleele sect ionedandsta inedwith phloroglucinol ant.!HCI fromtwo daysarte rantbl-sistofruitmaturity(Figure 4).

Thepedleelseeuene d twodaysdterantbesiswasdesign atedas week0in t'igur e 4.The valuesfortheproporti onorxylemtototal pedicelcross-section alareasror theproximaland distal portionsoreach pedicelwerechosenfromsectio ns approximately0.5mmfrom tht absc ission zon e.Theproport ion ofxylem was greatest in theproximalsegments with a meanor50%forallpedteels, and somewha t lessforthedistalsegments with a meanof40%.Corres pondingvalues forxylem at theabscissionzonefor the same pedicelsranged from4%to13% .

Xylem tissues ofthe leafabscission zone andthezoneof attachment of the pedi celwith thefruit werecomposed primarilyof fibr es and tracheids.Xylemof thepedicelextendedtothe pointor att achment ofthesepals and fruit where the

(33)

FIgure 1:A. Floralprimordia.Noxylem vesselsapparentB.Entire inflorescence.Traebearyelementsadvancing acropetnllytoward

nowerbud tips. Stained ...itb0.1%ehlorazolblnekEo

A ::116x B:: 116x

(34)

23

,I

' .

A

B

(35)

Figure %:A.Entireletlcreseee eewith tU fbear yelementsr('lfhin~

thetipofthe largestbud.B.Xylemtissue of thepediceland sepa lsofabud.Noabscissionlone is evident.Stained with

0.1%chloraaolblack E.

A"l1&x B=116x

(36)

25

A

(37)

Figure 3: A.Enlarg ement of traehearyelementsthroughthepedicel of a bud. Elementsare relativelyshort ,but noabsciss ion zone is evident.B.Wholepedicelillustratingdecreased xylem at the

abscissionzone. Stainedwith 0.1% chlorazol blackE.

A= 290x B..18 . 75>:

(38)

27

A

(39)

Figure 4.: Percent

o r

xylemrela tivetototalcross-sectionalarNl inthe proximal,distal,and abscissionnlsegmentsorpediccls

agedtwodaysalteranthesis(Oweeks)toeightweeks.

(40)

c

- ---:--- - ---- - - ----

...

, .:-- - ---

(41)

majorityofit extendedintothe sepals10form a continuousnetpntn-ruwith venation similartothai ofaIl'lIf[Figure5,1\).Amain mid-veinIornu-d and vessel elementsbranchedlnt('r nll ~'to the sepaledgeto connect withstm ndsof xvlom running alongthe~('plllllt'r iphl'r~" TIlt' venafionW:L~r0I11111.'\I',withno blindends.Approximately·1'2"[,or thesurtece:HI'a .,ftheelenn-dund~I:lilll'd sepals was xylemtissue.The sylomvasculatu reof the ovary.lind lan-rth,'fruit, wasI~!extensiverelativeto organsize.Smallfruit1< S milldiaUWIt'r l conta ined approximately26% xylem tissue per fruitdisk sUffar(' nrea.Xylem tissue comprised approximate ly11%ofthesurface area.ofclearednndstnincd disksof largertruitf>10mmdiameter ), Ttac hcary elements exte nded from the circleof xylrminthereceptacle tissue atthe fruit' s proximal point ofattac hment.

Themajo rityof itpassedthroughpedicel tissuedirectlytothe ovules.Rem aining branches ofthe xylemspreadalongthe fruit walli nthe proximalrruit half and thenbranchedfurtherintothe distal half.Typically,the fruitwallconta ined three tofivearms of xylemthat reached fromtheproximaltothedistalendof thefruit withinthefruitwall. Herethey converged8.!1twobundles into thestyle.

Alterfertilizati on,the ovary wallbegantoswellandthestyle excised,lea ving broken ends of xylem at thedist al endofthe fruit(Figur e5,B). Generally,the xylemof the fruit wallW8.!lcontinuous withno blind ends except for these two brokenbundles.The distalporti on of the fruit appeared to bepoorlysupplied withxylem relativeto theproximalhalf primaril y because themajority of the xylemvesselsof thefruit laywithin the placent al tissuewheretheybranched directlytothe ovules (Figure6,A),The xylem of theplacentawascom posed solelyofshortand crookedvessel members(Figure 6,B).

(42)

Figure 5:A.Xylempatternofthesepals.B.Longit udinal median section of pedicel,sepals,a.nd fruitillustrating xylem a.tthe distalportionofthe fruit.Age-oneweekafterfertilizat ion.

Stai nedwith0.1% ehlorazolblackE.

A :: lOx B" 75x

(43)

32

A

(44)

Figure 8:A.Median sectionof fruitillustr atingtbe concentrationorxylem inthe placentaltissue.B. Tracheary elemen tsoftbe placentaleadingto the ovules.Sta ined with

0.1% chloraeol blackE.

1\= 5.6)( ^Bs 116 )(

(45)

34

A

(46)

35 3.2. Dil!ltribution of Fluorescent Tracer

The pedi celxylem washeavil y sta inedwithberbe rine bemi-eulphet e and laterallea kageofthedyeresulted in allcells containing ligninto be stained.

Figure7 illust rates the extent01dye passage alongthe pediceland thereduct ion of xylemat the abscissionzone.Atthepointwhere the sepalsattachto the pedicel,thedye trave lledinto boththe sepals andtheCr uit(Figur e 8). The propor tion01berberineherni-sulphatethat enteredthefruit concentrated inthe pleA~_lltaltissueof the proximal half of the (ruit(Figure8, B).The mean fluorescence perunit surfaceareaofthe proximal fruit hairwas 28.S,whereas the mean ofthe distal fruithalt was13.9 (relative fluorescen ce units). The distal Iruit haircontainedless relat ive Iluoreseeuce per unitsuereeeareathan the proxima l fruit hilI!. SpotsoCdye were rarely seen in the fruit wall. No dye was observedin thetarblossomend of anyCruit . This patternotberberinedistribution was consistent in allsmalland largegreen fruit,but mature ripefruit showedDOdye uptake. Removal

or

the sepalsfrom arou ndthe fruit caused a significantly greateramountof dyeto travel into thefruit (meanrela tiveIlucr escence of63.6) comparedwithfruit withtheirsepalsintact(meanrelat iveIluc reeceace of42.6), (F = 16.61, p=0.0I5) .

3.3. Distributionor4SCa

Frui tusedinthefollowinganalyses were assignedto one of two classes:

thosethat weighed 0.05 g (30mm³)orless(hereinafterreferredto as-very small fruit-)and thosethatweighed morethan0.05g.

(47)

Flgure 71 Proximal (A) abscissional

(a)

and dista l(C)pedieel cross-sed ions illustr atingdecreasedxylemat theabscissionzone.

Stai ned with0.03%berbe rinebemi-sulphat e.

A" 13x ^{B "} 1J)( c '"1Jx

(48)

37

(49)

FIgure 8: A.Cross-sec t ionofsepalillustrat ingxylem vessels.

B.Cress-sectionoftheproximalendof thefruit.Yellowspots indicat eberberine-stainedxylem.Stainedwit h 0.03%berbe r i ne

hemi- sulphate.

lOOx B"~, 6 x

(50)

39

A

(51)

·10

The]('3 \' C:;(n=13),inflorescences (n=I &),andhuil(n=5.j) ou tom at o plant

sternsdiHNenl iAllyaccumula tedcalcium (TableI).The fIldiollctiv it},co untedin the](' [\\" (' 8 ,[thegreat estaceum u lntors].was 508timesgr e at erthnntill'dist:al !lntr of fruitweighingmort' tha n 0.05gfw,hweight.Whol(' fruitweighingIt''''sthan 0.01g(n=8),accumnlnte dsignlficanflymor e cnleiumper fff·sh wt'ighll-t.2xI06) thandidmediu m andlar gerlrui t ranging from O.Sg-7.5g (2.:lxlOr.,n=·16), (F~68.58,,<0.001).

Ana lysisofvaria nceof thenine plantpar t groups shewcd asignilic a nt dillerence in calciumaccumula.tio nbetween leaves andallothertlsaues (F= 1.15.

p<O.OO l ). Fruit greate r than 0.5 g accumulate dthe least calcium (F= 37.lg, p<O.OO l );othermaximum nonsign illcantranges areindicatedin Tablet, Proxim alhalvesoffruitalwaysccnutncdmore calcium thandistalhalves,but the differencewasnot signiricant (F=4.05,p=O.06).

Ca lcium uptakeinto the fruitwasdependantODthe totalwaterintakeby thefruit (increas eof volumeplustreuspir eticualloee]overthe fourday experim entalperiod(Figureg). Regressi onenelyeis on0= 30fruitgavethe followingequation:

y~-4.74xlo'

+

3.2'x lO' (Xj

Y=radioact ivit y ofcalci u mandX

=

tota l water intake

The regressionco efficientisr

=

0.89

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Plant

"arls

"

T.ble I: MeantSCa dpm valuesofplantparts.

Mean MaximumNODsipificant

Caldum(d pml Rangn eX!Is.e .m.foll

,....r

St"(llll Flower Dud F1ower Whol{'rr uit <O,Olg Pedice l Petiole Proximal Frui t Half DistalFruitHaIr

3.2xl07!1. 7xI07 (13)

;.5xl~6.;IIOs(5-11 7.4 xl06! 1.8xIO&(11) 7.4x 1~1.3x106(4) 4.2x I06~ 1. Ix l06(8 ) 3.Ix I06!1.5x lOS(54) 3. h. J06~3 .6xlOS(4) 1.6xI06.!5.8xIO~(46) 6.3 xlOt.!2.5xIO^t(46)

A D B B C C C D D

(53)

42

Fruit with a large volumeincreaseaccumulat ed sign irieantly morecalcium over the experimental period(F=1l3.0,p<O.OOl).

Calcium accumulationcomparedtothefruit's increaseinvolum e was constantlor all fruit grea terthan 30 mm3in volumeand 0.05gin freshweight.

However,Iruit em afler than 30 mm3or 0.05gaccumulat ed,onavera ge, 7.5times thecalcium perincreaseinvolum e than did fruitlarger than 30 mm 3.ThemeaD calcium accumulationrelativetogrowth fota very small fru it wasl.lxlO4 dpm/volume increase comparedtothe const ant valueof 1.3xIWdpm/vollime change(Otfruit between0.5g and 7.5g or325 mm3and 6600 mm3tC5pcctivcly.

Logari thmi ctran sform ati on of the Xaxis variable allowed anyvariabili ty of calciumcontentamong very small Cruittobe visuallyobvious.Figu re 10 illustrat esthe differen cebetween calcium accumulati onrelativetovolume increasefor fruitlesstban30 mm 3compa re d withhuit largertha.n30 mm3.

Transpirati onallosses perunitIruit surfaceareawerealsogreatesllorvcr), small Iruit «30 mm3)wherecuticular eon duete uee was0.18mg/h compa redto themeancuticular conduct ance010.002mg/bforIruitrangingfrom 321)mm3to 6600 mm3 (Figure 11).Fruit lessthan30 mm3 sho wed an exponenti a l wat erlOS'!

perunit area that avera gedDOtimesmorethanlarger fruithom325mm3to6600 mm3.Fruit between 30 mm3and 321)mm3 sbowedan intermediat erate01water loss. The mean transpir ationrateor0.002 mg/ hlortbe largerfruit representsa constantrate01water lossper unitsurfacearealorall theseIruit.

Overtbe lourday experimentalperiod ,theincrease01Iruitvolumerelative

(54)

Fig ure 11: Callum(dpm)r-elat iveto total rr uit water intake overthefourJayexperimentalperiod,

(55)

y,.-47 43 .. 32 9 X

TOlaIWaler lnl.ke~ Fruillmg)

""•\fJl)

.

(56)

Figure 101 Calcium (dpm)rela t ivetogrowth over thefourday exper im ental per iodfor all fruits tested.

(57)

46

. ^.; ^.. ^.". ..

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.. .",-- - - -- - - - -- - ----,

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I

^ro

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FluIt_.,.,...·

(58)

F1su re 11: Cuticularecedueteeeeor rruitrelativeto theirsurrace .reL

(59)

..

+ i

₀₂

1 .i I ^..

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! _'" -._-

i

(60)

, ••

toinitia lvolumeW&!lgrea testror(ruit lessthan30mm3 andWA.97 timesgreater thanmediumand large sized fruitranging from325 mm!to 6600 mm3,The meanrelativegrowth increase of very small fruit«30 mm3)was3.6xl0· 1gover the experimental time,whereasthemeangrowthincreeae of fruit (rom 325 mm!

to 6600mm!WAS5,lJxl O·2goverthesame period(Table2).Loga rithmic transform ation

or

fruitvolumeillustr atesthata const antslope exists

r or

the relatio nship betw een rela tive increase

or

volume and f,uitvolume(0=54),(Figure 12):

Y=relat iveincrease

o r

volumeandX=fruit volume Theregressioncoefficientisr=o.gg

Thetotalamountof water cntering anexpa ndi ngfr uitequals the increasein volume plus tr anspira tic nallosses. Theamount

o r

^calciumin eac h fruit rela.tive 10itstot alwateraccumul at ionoverthefour day experimental periodwas independen t offruitvolume. Theratio ofcalciumto wat er inta ke was consistent fotall fruit sizes.Verysmallfruit ,lessthan 30 mm³,did accumulateless calcium pertotal water influxthanlarger fruit butthiswasnot asignificant differe nce (Table2)-

The grow th and amount of calciumaccumulatedby afruit wa.salso depende ntonits position ona truss,The first (most proximal or closesttothe main plant ste m) fruit on atruss, accumulatedsignificantl y more calcium than

(61)

50

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Figure 12: Relative volumeincreasecomparedto initialfruit volume.

(63)

"

03

52

-""7"""- -- - -- - - --- - ---

Y

=

0.398 -O.10110 0.0X

00 .--~~-.-~~ ----,-~_..•-

~ 00 ~5 10 15 ZO ~ W Fruil Volume. loolOmm3

(64)

53

themost distal fruit ofthesame truss (F= 13,92 p=O.Ol). TheIruitin between these two extremes accumulated less calciumwitheachposition away from the

firstfruit. The amountof calciumaccumulatedisrelatedtotheCruit position

(Figure13):

Y=2.15xIOs•2.7Oxl04X

Y

=

rsdioaetivityofcalciumand X=trussposition

Theregression coefficientisr=0.81

The aboveequat ion was for asingle trusswitheight fruit. Variability in dprn counts betweentrussescbangedthevalue01they-iute rcept ,however,the linear relationship remained the same.

Asimilar relationshipexistedfot the increase in volumeoverthe experimental period andfruitpositiontorthesametruss ofeightfruit (Figure14):

Y=increasein volumeandX=trussposition

The regressioncoefficientisr=0.82

The change involumeofthe firstfruit was significantlygreaterthan all others ofthe truss and declinedtothe most distal fruit(F=15.34,p=O.OO8).

However^Ias a proportionof growthrelative to size atthe beginningoftbe

(65)

Figure 13: Calciumcontent relative tofruit positionAlong thetruss.Fruit#1 isthemostproximal.

(66)

''(1 _._ .

o

55

FruilPositiononTruss

(67)

Flgu re 14: Increase orvolume relativetorruitpositionalong thetruss.Fruit#1is themostproximal.

(68)

57

FruitPl:llitiononTruss

(69)

5 "

experim ent,the cbange involum e was relativelyCODstant for all fruitof thelrUS9

largertban30mm3or0.05 g.These verysmall fruitgrewthefastest com pared toothersonthe truss even though they werethe most distalfromtheplant stem (Figur eIS).The proportional growth of the remaining fruit was approxima tely equal,regardl essoftheir trussposition.FigureISillustrat es the generalhendof increase in volume relat ive to initialsizeIcr thedinere nt fruit alongtbe truss.

Data of tbecalciumconte ntand volum eincrease of theremainingfruitof trusses are listed in Appendix E.

TheIru it epidermal surfacelacked stomata ,butstomatawerepresent on both surfacesof leaves andsepals.The abaxialsurfaceof theleaves contained.the highestmean stomatal density0145/m m2 andanadaxialmean 0IO/ mm2.The meanDumbe rorstomataonthe abaxialsideorthesepalswas24/mm²and fJ/mm²ODtheadaxialside.Pedlcelscont ainedII.meanof3/mm²on their epidermalsurfaces.

(70)

Figu re 151Relativegrowth orrruit comparedtotheir positio nalong the truss. Fruit #1lsthemostproximal .

(71)

60

ooo\-o-~-~-~- Fruit Po5iliOnon Truss

(72)

fil

Chapter 4 Discussion

Thedevelopment

o r

xylemtissue within the primo rd iaof the tomato inflorescence throughtofruit malurity affectsthe modeof waterand calcium supply to thepedicel,sepals,andfruit. Theextent

o r

vascularizationwithinthese organsplus effects01 transpirationdetermine the site'Swhich accumulate calcium.

Theinflorescence01thetomato differentiatesfrom thevegetativeapex and developsin the mannerdetailed by Sawhneyand Greyson(1072). Atflower maturit y,a complete ring

or

xylem vasculatu reextendsthrough the pedicelto the recept acle,whereitbranc hestothe sepals,pet als, ant hers,and theovarytissues.

Anabscissio nzonein themiddle or the pedicelisindicated by adramati c decli ne inxylemtissuecross-sectionalarea.Thedat apresentedhere show thatthe abscission zone xylem is composedofshort,irregular vessel mem bers tha tpers ist fortheduration of fruit development . Withi nthe abscissio nzone,vessel members,tracheids, andFibers ofthemetaxylem andsecondaryxylem railto develop as theydo intheproximalanddistal portions on eitherside orthe abscission zone.Jensen and Val dovinos(lg67) describe d the anatomyor the abscissionzone ofthetomato flower atanthesisand not edthat vascular tissueis con tinuousthroughthezone,butdid not describe thexylemtissue oritsrelativ e

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62

amounts with in the ped icel.Hudak(lOS7)no ted tha t the xylem in theIlbsl'is.~ion loneWlLS sigIljfieantlyless thantheproximal and dist al pedicelportions.The xylem patt ern wascompa rab leto tha.tof thepedicelofthe man go(Bar nell1039) in wh ich the amount ofxylem wasdecreased andthecylinderof vascular tissue wasbrokenintoanirreg ular ringofxylemst ra ndsat the abscissionzone. Baird and Webst er(107Q) notedthatxylemvessels are unusuall y smallorabsent from the abscissionzonesof mostfruit andconside red thetoneas aregion ofabrupt structu ral transition .AJoni(IOS7) studied the irregul arvascular attachmentsite betweenthe xylem ofII.leafandstemof the palmRhapiscue/sa .Atthenode betweentheleaf and plant ste m, the vessel syslemof thelear connectstothe vessels ofthestemvianarr ow traehelds. Thetracb cldsin the noderegion presu mab lyprotectthe vessels ofthe ste m from cav it at ion when the leafdrops ell.Lee(19g9) proposed lhatthe abscissionzonein thetomato pedicel resists water now throughthexylemand transfersthedem andforwalerby the fruit to thepbloem, thuslinking water anddry matterinfluxto the fruit.

Gene rally, the presence ofaIluor eseentdye suc h as berb eri nehemi- sulph ate,can be detectedinsmallerquan tit ies than can non-flu orescent dyes (O' Brien and McCull y1991), makinganywaterpassage through the xylem obvious.Iftranspir a tional tensionsare high orsue lionisapplied tothe endof the stem,berbe rine herni-sulpha teflows withthewaterpathways and thereforewill indicat efundion alwater- conducti ngvesselsandtracheids(Dixon and Peterson 199Q). Genera lly, berberine stainsligninandsuber in in plant tissues (Drundrettd al.Iggg).However, the dyetendstobleedlaterallythroug h vesselelements and

(74)

st nincellsin which the dyewasnotear-led,making quan tificationofwat er- conductingvesselsandtracheidsimpossible.In everysection,xylem vessels and trnehcidswer estained pillsanycells containing lignin (Figure7),Thetechnique (,'st:l.i"ing Iu net lonnlxylomis presentlybeingrefined(peterson,pers.comm.]

Ik rlll'rinehe mi-sulphateispreferable to aphloroglucinolandlIelstainbecause thelatt ersinlOSIoravnrietyorpolysaccharidesbesides lignin,whereasthe presenceorberberineillustrat es areasorwaternow-thedye sta ins lignin whereverittravelswith thewater.Therefore.berberi ne stainin gis usefulto show npoplast iccontinuity throughthelengthoftbepedicel.

Thexylemtissueorthefruitconcentrated withintheplacent alregion.

These tr achearyelements were stainedwith berberinebecause ofwatermovement to theovules.Eventhough xylem existedinthe dist alfruitportions,dyedid not passtotheblossom-end ofthefruit.Thevesselseith er did notfunctionaswat er- conducti ngpassageways or, moreprobably,tra nspirational waterlossesIromthe fr uitwereinsurricient todrawdye tothedistalend, Asthefru itsurfacelacks stomata,transpirationmustbe cuticular,Asignificantl ygreateramou ntof dye travelled intothefru it when sepals wereremovedbeforetheste m endswere immersedin dye.Thissupports theideath at transpirational pulland amountor water lossfromthe sepalsdivertswateranddyeintothe sepalsratherthan allowing itto continuefromthepedicel into tbe fruit.wiersum(1066) immersed tomatotrusses intoasolutioncontainingthedye,Light Green,The dye travelled apopla.stically, staining thevasculature ofthe pediceland calyx butabru ptly endedwithinthetissueorthe recepta cle.Dyewas rarelydetectedin veins of tbe

(75)

..

(ruit wall.Wiersumdid not confirmwheth eror notthemaj ority of dyeflowed throughthe plac enta l tissueto the ovules.The results of thisthesis sup port the viewthat little apoplasti cwaternowappearstoenter the fruit.In these experiments, thepresence of berberineheml-sutpb ete correla ted with the observa tionof greatest xylemconcentration withi ntheplacentaltissue.

Ehretand Ho(lOSSa)have shown thatthehydrauli c conduc tance

or

xylem sap diners among thepedicel,pedicel-fruitjunction, and (ruit tissues.Pressurized waterforcedthroughthe xylemtrachealY elementsshowe darestri ctiontoxylem nowthatincreasedin thesequenceof pedicel

<

pedicel-fruitjunction

<

fruit. A lowhydraulic conducta nce implies either a lowamountofxylem wat ertransport , some form of restri ction tonowwithin the vessels,or fewvessels totransport water.The fruitcont ains lessxylemper cross-sectionalareathan docsthe pedicel, and hasa lowtranspir ation rate,thereforeitmay hespeculated that waterwithinthe pedicelis primarilyattractedto the sepalsthat exhibit a high transpirati onrat e. Measurementsofwater pot entialgradients wit hin the tomato plant showed that aresistan cetowater now existsbetweenthepedicel and tomatofruit (Leed al.1989).This observation maypartlybethe consequenceof the short,crookedvessel memb ers thatcharacte rizethe xylemvasculatureof the pedicel-Irui tjunct ion.However,itisnot expectedthatvessel members with simple perforationplat eswould greatly impedewaternow.

The organsof thetoma toplantaccumulateradioa ct ive calciumtodifferent extents.Leaves contai ned morelabeled calciumper wetweight than all other plantpartsexamined. Developingandmature leaves cont ainedagreate r

(76)

proportion ofxylemv('$.<;l' 1strands than didflowersorfruitandwerewellsupplied withetometeon both sides. Thesecharueteristicswould cont ributeto ahigh trnn~pirat ionnl('npltrity,therebydrawing calcium intothe leafin the tm nspirntiunstrea m.Armstrong and Kirkh yOgjOI notedthat tomato plants grownina.highhumidity environment(Q5t:'V RJI.), showed a markeddirrerencein calciumdistribution relati ve to plantsin a lowerhumidity (50%R.II.).In high humidity ,the level ofcalciu m in the young leaveswasverylow andcalcium accumula tedin thestems .Therefore,duringperiods of highhumidity and hence low transpiration, calcium tendedto accumulat ein stems insteadof leaves.

Whole flowers andbu ds, bothof whichhave intact sepals, andsepals taken separately,allaccumulatedrelativelylarge amountsofcalcium.Thiswas expected , since sepals havestomataon both surfaces and arewellsupplied with xylem vessels.

The pedicclaa.nd petiolesaccumulated less calciumthandid leaves,sepals, buds,andIlcwere. Theseorgansrepresent vascularpassageways and the occuranceof calciumin thepedicela and pet iolesisdependen tontranspira.tional pullofxylem water pluscalcium exchange on negatively-cha rgedsites of the xylemvesselsurface(see Hanson1084).Because oflateralleakagefrom xylem vessels(seeHangerIlli g), some calciumwas absorbedby tbe pedicclsand petioles.

Considerably morecalcium was expectedto accumulatein organs whicb mainta ined agreater transp ira.tional pullon tbe xylemwater.

Thetomato fruit containedthesma llestamount ofcalciumperwetweight

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66

rclaliveto all otherplantpartssamp led.Thispatter nWl\Sconsistentfor1111 rc pl ieate trussesRodwas correlatedto therel ative amounts

or

xylrmvaseulntu re.

Asptevlnuvlydeserlbcd.huH arf'poorly supplied wit hxylem rvlative topl'lIi<'l'1, sopnl, and lent.The proximalfruithalfronsislt'ut!)' eontnlnodmon- cnh-iumpN unit wet weightthanthedistal half(Tllblt'I). In a studyinvolvingr-nleium dist ributionwithinplantsgro wnat various salinities,Ehretanti 110(HI~lIhl:l.1~1 observed alowercalcium concentrationin thedisla l hair

o r

thc Iruit rl'lalivt· to the proximal half.Theynotedthat removal ofsepals producedIIIlinerensed inci denceorHER andsugges tedtha t the sepa lsdraw water to wardtherrutt becaus e oftheir high transpirationalcxpneil.ies.However,th e presen t rt'llult!l sh ow thatsepal removalres ults in an increas ednow(Ifdye intothe fruit Sep al transpirationalpullenhanceswate rnowacrossthe pedicel, andwater nowwould be diverted into the sepalsrather thaninlothefruit.

DevelopingFlower buds, flowers,andfruit receivesign ificantlylesscalcium thanmatu re leaves or the samepla n t.Ithas previouslybeenrecogn ized that calciumisnot significantly transloc at ed fromoldertissuestoyoungerplant parts evenundercalciumstressconditions (seeHan gerI1no).Whencalciu mis abundantin thesap, the distributionoftheion willbe closely relatedto theratc oftranspirationandcalciumwillmove prima rilyinto transpiringleaves(see Clarkson 1984). Onceitenterstheled,muc hof the calciumalJsorb<'d byle af cellswill beboundby oxalicacid,generated during nitr atereduction.Allplic atiun of calciumtothe soilandto thefoliage orplantsdocs notin crease calcium concentrationinthefruits,because itbecomesboundas calciu m oxa.late dueto

(78)

thehigh oxalic acid con lent or leavesand sterns (EvansandTroxlerHI53) . Grow t hin organs suc h,ISfruit s and transpiringleaves influencecalcium distribution. W!wnI.u g(' quant.iticsofcalciumareabsorbedthro ugh thexylem of tI)('ro...l,theconeentrntion difft'fenlialbet...ecn loavesanrl Iruit isminim al, 1I11h"lIgh therat ioehangesdiu r nally.Calcium no...stotra nspiri n gleavesheeausc or ur-gatlvetrnnspiratl onalpull duringthe day,andtofruits andmeristerntissues III night whenthepullof trnns pirationdecreas es androot pressur eincr eas es.

Organs withlow trans pirationrates,such&3meristemsand fruit , accum u late need edcalcium (rom xylemsa p deliveredbyroot press ure duringthenight fllradrirldand GuttridgeIg84).However,an alternati ve exists to acquiring calcium.Growingpoint s, suchasmeristemsand fruit,undergonew wallsynthesis nndtherebycreate epoplastlc bindingsites (ma inlycell wallcarboxyl groups)that rjmreleasethecalciumboundtoexch a ngesitesODthex)'lem vesselwall s(see Clllr ksonW84).Thismayexplainwhy prefere ntialca lcium tran sportin thedark periodintomeristernsandfruitisDot limitedtointact plantswith root pressure but isalsoobservedinisola.tedshoots althoughat&lower levelofcalciuminflux.

Thedistribution ofcalcium usuallyla v ersleaves butinlowtranspiration environments , thcdiffe rentialbetween leavesandIruitisless(van deGeijn and Smc u klcts 10SI).

Fruitgenerally contain less calcium than leaves.Tomatofr uitco nt ain a higher percentage of citricacidin theircellsap tban stemsand leaves.Organic acid s generatedinthe respirationof youngshoo ts wouldbeut ilized in the synth esisof proteins,whereasfruit have a lowprotein synthesisand thereforelow