Developmental Biology and Induction of Phi Thickenings by Abiotic Stress in Roots of the ...

17 Pages·2017·2.96 MB·English

plants
Article
Developmental Biology and Induction of Phi
Thickenings by Abiotic Stress in Roots of
the Brassicaceae
MaketalenaAleamotu’a,Yu-TingTai ID,DavidW.McCurdy ID andDavidA.Collings*
SchoolofEnvironmentalandLifeSciences,TheUniversityofNewcastle,Callaghan,NSW2308,Australia;
[email protected](M.A.);[email protected](Y.-T.T.);
[email protected](D.W.M.)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:25May2018;Accepted:17June2018;Published:19June2018
Abstract: Phithickeningsarespecializedbandsofsecondarywalldepositedaroundradialwalls
ofrootcorticalcells. ThesestructureshavebeenreportedinvariousspeciesfromtheBrassicaceae,
including Brassica oleracea, where previous reports using hydroponics indicated that they can be
inducedbyexposuretosalt. Usingrootsgrownonagarplates,weshowthatbothsaltandsucrose
caninducetheformationofphithickeningsinadiverserangeofspecieswithintheBrassicaceae.
WithinthegenusBrassica,bothB.oleraceaandB.napusdemonstratedtheformationofphithickenings,
butinastronglycultivar-specificmanner. Confocalmicroscopyofphithickeningsshowedthatthey
form a complex network of reinforcement surrounding the inner root cortex, and that a delicate,
reticulate network of secondary wall deposition can also variously form on the inner face of the
cortical cell layer with phi thickenings adjacent to the endodermal layer. Results presented here
indicate that phi thickenings can be induced in response to salt and water stress and that wide
variationoccursintheseresponsesevenwithinthesamespecies.
Keywords: phithickening;secondarywall;Brassicaceaeroots;Brassicaoleracea;Brassicanapus;salt
stress;waterstress
1. Introduction
Phithickeningsarepeculiarsecondarycellwallstructuresfoundinthecortexofplantrootsin
diversespeciesrangingfromgymnospermstoangiospermsandfrommonocotstoeudicots.Theywere
firstreportedintransversesectionsintheringofcellssurroundingtheoutsideoftheendodermisin
rootsofTaxusbaccatabyVanTieghem[1],whodescribedthecellwallthickeningswiththeFrenchterm
“reseausus-endodermique”whichmightbetranslatedto“super-endodermal”or“peri-endodermal”
network. ObservationsoftherootsofseveralRosaceaespeciesbyRussowin1875showedsimilar
coordinatedwallthickeningsinadjacentcorticalcellsthatresembledtheGreekletterΦ(phi),andthis
leadtotheadoptionofthename“phithickening”[2]todescribelocalisedwallthickeningsseenin
corticalcellsofroots. Whilesimilarstructureshavenowbeenobservedinmanyspecies,webroaden
thedefinitionofphithickeningstoincludespecialised,reticulate,orlocalisedband-likesecondary
thickenings that form only around the cell wall of root cortical cells, rather than across the entire
wallsurface. Thus,ourdefinitionwouldalsoincludethemorecomplexwallthickeningsintheroots
of some epiphytic orchids, a series of structures sometimes referred to as a ‘pseudovelamen’ [3,4].
Another related type of cell wall thickening in the root cortex has been described as a ‘crescent
thickening’ because the thickening is limited to the inner faces and sides of the cortical cells [5,6].
Thesefindingsconfirmthatmultipletypesofsecondarycellwallthickeningsarepresentinthecortical
cellsofplantrootsandwouldargueforourmorecomprehensivedefinitionofphithickenings.
Plants2018,7,47;doi:10.3390/plants7020047 www.mdpi.com/journal/plants
Plants2018,7,47 2of17
Intheearlyliterature[7],phithickeningswereclassifiedintothreetypesbasedonthelocation
ofphicelllayers(thelayerofrootcortexinwhichthephithickeningsarepresent). Aphicelllayer
locatedintheinnermostlayerofcortexadjacenttotheendodermisisdefinedasTypeI,anditwasthis
organisationthatVanTieghemdescribedas“sus-endodermique”. TypeIIthickeningsoccurwhenthe
phicelllayerislocatedintheoutermostlayerofthecortexadjacenttotheepidermis,anorganisation
describedbyVanTieghemas“sous-épidermique”orsub-epidermal,whileTypeIIIdefinesphicell
layersintheintermediatecortex,eitherinsingleormultiplelayers. VanTieghem[7]observedthatitis
notcommontohavemorethanonetypeofphithickeninginthesameroot. Thisclassificationisstill
usedtodifferentiatethevariouslocationsofphithickeningsinroots.
Although phi thickenings were first described in the 19th century, relatively little is known
about the function(s) they might perform in plant roots. One early suggestion for the role of phi
thickeningswasthattheymightplayasimilarroletotheCasparianstripbyregulatingsoluteuptake[8].
Both phi thickenings and Casparian strips are cell wall thickenings reinforced with impermeable
lignin polymers, and they are found in root cortical cells of some species and endodermal cells of
mostspecies,respectively. Casparianstripsaretypicallyimpregnatedwiththewaxsuberinwhereas
phithickeningscontainlittleifanysuberin[9,10]. Thelackofsuberininphithickeningsneednot
meanthatthesestructuresdonotfunctionintheregulationoftransportregulation,assuberin-free
mutantsinArabidopsisretainafunctionalCasparianstripthatcanblockthemovementofapoplastic
tracerdyes[11]. However,similardyeuptakestudiesinbothappleandgeraniumrootsshowedthat
dyemovementwasblockedbytheendodermisbutnotthephithickenings[12]. Anotherproposed
functionofphithickeningsisthattheyprovidemechanicalsupportfortherootcortex,andthatthey
mayactasreinforcingstructuresfortherootcortex[13]. Interestingly,Melville,etal.[14]proposed
thattheymightactasaphysicalbarrieragainstpenetrationbyfungalhyphae. Amorerecentstudy
suggestedthatphicellsmayplayapartinactiveuptakeofcationsandanionsforaccumulationin
vacuoles[15]. Allthesesuggestions,however,havenotbeentestedindetail,anditremainsentirely
feasiblethatphithickeningsperformmultipleanddifferentrolesinrootsofdifferentspecies.
Phithickeningshavebeenidentifiedinawidevarietyofplantspecies,butnosystematicsearchfor
thepresenceofphithickeningswithinagivenplantfamilyhasbeenconductedsinceaseriesofstudies
byVanTeigheminthe1880s. Consequently,ourcurrentstudyprovidesasurveyofphithickening
developmentintheBrassicaceaeandalsoassessestheresponseofthespeciestosalt-inducedwater
stress.Weselectedthisfamilybecause19thcenturyreportssuggestthatphithickeningsarewidespread
withintheBrassicaceae[16,17](althoughtheydonot,apparently,occurinArabidopsisthaliana)and
becausephithickeningscanbeinducedinBrassicaoleracearootsby14-dhydroponictreatmentwith
salt[15,18]. Byanalysisof5-d-oldseedlingsgrownonnutrientagarplatesinthepresenceorabsence
ofaddedsalt,wedocumentconsiderablevariationwithintheBrassicaceaeinnotonlythepresence
of phi thickenings but also their response to salt stress. However, induction also occurred under
sucrose-inducedwaterstressinconditionsthatdidnotinhibitrootelongation,thusdemonstratingthat
rootgrowthinhibitionandphithickeninginductionarenotdirectlylinked. Wealsoreportvariations
intheabilitytoformphithickeningswithintwospecies,B.oleraceaandB.napus,whereonlysome
cultivars induce phi thickenings strongly in response to salt. Using confocal microscopy and 3D
reconstructions,wealsoshowthatphithickeningsformacontinuous,lignifiedringaroundtheinner
cortexofsomeBrassicaroots,immediatelyoutsidetheendodermis,andmoreintriguingly,thatthese
thickeningsoftenextendtoformadelicate,reticulatenetworkofsecondarywallmaterialalongthe
innerfaceofthesecellsadjacenttotheendodermallayer. Collectively,theseobservationsrevealthe
diversenatureofphithickeningdevelopmentintheBrassicaceae,andthatvariationscanoccurinphi
thickeningresponsestoabioticstressnotjustwithinafamilybutevenwithinasinglespecies.
Plants2018,7,47 3of17
2. Results
2.1. PhiThickeningsResponsetoVaryingSaltandSucroseConcentrations
Following previous work in B. oleracea in which 80 mM salt treatment administered through
hydroponicsinducedphithickenings[15,18],weconfirmedthatsalttreatmentcaninducetheformation
ofphithickeningsincultivar‘MarathonF1’. Inourexperiments,wereplacedhydroponics[15]with
growth of seedlings for 4 d on agar plates containing 1/2 MS salts or 1/2 MS salts supplemented
with 80 mM NaCl. Whole roots were fixed, cleared with potassium hydroxide, and stained for
ligninusingbasicfuchsin[16,19]. Comparedtosalt-freecontrols,whichonlyrarelycontainedphi
thickenings(Figure1a),80mMNaClinducedstrongdevelopmentofphithickeningsimmediately
outsidethecentralstele(Figure1b). Usingimagesderivedfromafluorescencedissectingmicroscope,
we developed a scoring system based on counts of the number of induced phi cells (i.e., cortical
cells containing phi thickenings) within a set length of cleared root. We used this system to score
the response of phi thickenings to varying salt (Figure 1c) and sucrose concentrations (Figure 1d).
Theresponsecurvetosaltstressemphasizedthatthedegreeofphithickeningformationincreased
withsaltconcentration,andthisincreasewasaccompaniedbyreducedrootelongation(Figure1c).
Importantly,inductionofphithickeningswasnotspecificallyaresponsetosaltstress,sinceinduction
wasalsoseeninresponsetosucrose(Figure1d). Furthermore,whereassalt-inducedphithickenings
increasedasrootelongationdecreased,thisresponsewasnotseenwithsucrose,whererootelongation
wasnotsignificantlydifferentatanintermediatesucroseconcentration(2%)atwhichstronginduction
of phi thickenings occurred. Therefore, we conclude from this analysis that root elongation is not
correlatedwithphithickeningformation,whichsuggeststhatphithickeningsplayaroleotherthan
growthinroots.
2.2. SurveyofPhiThickeningDevelopmentintheBrassicaceae
WeextendedthesurveyofphithickeningformationindifferentspeciesfromtheBrassicaceae
conductedbyVanTieghem[16]byinvestigatingtheinductionofphithickeningsbysalt. Wealso
surveyedthesalt-inductionofphithickeningsindifferentcultivarsofbothB.oleraceaandB.napus.
WithinthesevenmajorgroupsofB.oleracea,andwithinthe20cultivarstested,allexceptonecontained
phi thickenings to some extent (Table 1). The cultivars in which phi thickenings were abundantly
inducedweremainlytheItalicagroupofB.oleracea(broccoli),whereastheGongylodesandBotrytis
groups (kohlrabi and cauliflower, respectively) typically had fewer phi thickenings, and with the
exception of Romanesco, were not strongly induced by salt. We also measured root length of the
seedlingsanddeterminedhowtheinclusionof80mMNaClaffectedrootgrowth. Asshownbythe
rootlengthratio(Table1),somecultivarsweremoreresistanttotheeffectsofsaltthanothers.
Within other species of Brassica, similar variations in phi thickening induction were also
observed (Table 2). Within the five canola cultivars of B. napus surveyed, major variation was
seen, whereby the three spring cultivars ‘Hyola97CL’, Canola ‘Hyola474CL’, and Canola ‘Archer’
showedstrongly-inducedphithickenings,whereasthetwowintercanolacultivarstested,‘Edimax’
and‘Sensation’,eithermildlyorcompletelylackedinducedphithickenings,respectively. Inother
Brassicaspecies,however,withtheexceptionofoccasionalthickeningspresentintheB.rapasubspecies
Pekinesis‘Wongbok’,nosubstantialinductionwasobserved(Table2). Inconclusion,theinduction
ofphithickeningsinthedifferentBrassicaspecieswasshowntobestronglydependentonspecies
andcultivar.
We also surveyed phi thickenings and salt induction within other genera of the Brassicaceae.
Contrarytopreviousobservations[16],wefoundthatphithickeningsweregenerallyrareinother
genera,andtheywerenottypicallyinducedbysalttreatments(Table3). Extensivephithickenings
wereseeninSinapsisalba(whitemustard)inbothsalt-freeand80mMNaClconditions,withthehigher
phicellscoresattributedtothecomparativelyshortlengthofcellspresentwithintheinnercortexof
thisspecies,whichprovidedmorecellswithinthedefinedmeasurementlength. Thlaspicaerulescens
Plants2018,7,47 4of17
(alpinepennycress)alsoshowedhighlevelsofphithickeninginductioninbothsalt-freeand80mM
NPlaanCtsl 2c0o1n8,d 7i,t ixo FnOs.R POEtEhRe rREsVpeIEcWies in which phi thickenings were observed included Iberis 4a mofa 1ra7
(candytuft) and Lobularia maritima (alyssum) that showed strong, salt-induced formation of phi
amara (candytuft) and Lobularia maritima (alyssum) that showed strong, salt-induced formation of phi
thickeningsequivalenttothoseoftheBrassicaspecies.
thickenings equivalent to those of the Brassica species.

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sucrose.Scoringinvolvedcountinginducedcellswithinasetlengthofroot(leftaxis,dottedlines)and
or sucrose. Scoring involved counting induced cells within a set length of root (left axis, dotted lines)
wasnotrelatedtorootelongation(rightaxis,solidredlines).Dataaremeans±SEM,n>21plantsfor
and was not related to root elongation (right axis, solid red lines). Data are means ± SEM, n > 21 plants
eachdatapoint.
for each data point.
Plants2018,7,47 5of17
Table1.PhithickeninginductionbysaltindifferentBrassicaoleraceagroupsandcultivars.
RootLength(cm) RootLengthRatio PhiThickeningScore
Group Cultivar SourceCode Measurement(Days)
0mM 80mM (80mM/0mM) 0mM 80mM
Acephala
‘Dwarfgreencurled’ 1 10 1.9±0.3 2.2±0.6 1.09 0 19
kale,collardgreens
Alboglabra
‘Kailaan’ 2 5 3.2±0.6 3.4±0.5 1.06 8 21
Chinesebroccoli
Gemmifera ‘Eveshamspecial’ 1 10 4.9±1.2 8.5±0.1 1.73 21 25
Brusselssprouts ‘LongIslandimproved’ 3 6 3.4±0.3 3.0 0.86 0 10
Gongylodes ‘WhiteVienna’ 4 13 2.4±1.2 1.5±0.5 0.62 0 6
kohlrabi ‘PurpleVienna’ 4 5 3.7±0.8 2.2±0.4 0.59 0 1
‘Mammothredrock’ 4 5 4.9±1 4.6±0.8 0.94 3 45
Capitata
‘Goldenacre’ 4 6 4.3±0.4 3.5±0.5 0.80 0 2
cabbage
‘RubyballF1’ 1 5 5.1±0.4 4.6±0.2 0.90 18 43
Botrytis ‘Quickheart’ 1 5 4.4±0.2 3.1±0.4 0.70 1 15
cauliflower, ‘Phenomenalearly’ 2 5 4.9±0.4 3.1±0.4 0.64 3 0
broccoflower, ‘Snowball’ 2 11 1.0±0.2 1.0±0.2 0.99 14 15
Romanescobroccoli ‘Romanesco’ 3 6 4.3 3.0±1.5 0.69 4 47
‘MarathonF1’ 1 5 4.8±0.6 4.1±0.2 0.86 21 39
‘Calabrese’ 3 6 3.3±0.8 4.1±0.02 1.25 33 43
‘DiCiccio’ 3 11 2.3±1.1 0.9±0.1 0.40 8 20
Italica
‘Belstar’ 3 6 3.4±0.4 2.7±0.5 0.79 18 40
broccoli
‘Umpqua’ 5 5 2.9±0.7 3.2±1.0 1.10 22 39
‘Goliath’ 5 5 4.4±0.7 4.2±0.3 0.95 25 46
‘Summergreen’ 2 6 3.7±1.1 3.8±0.3 1.03 6 31
Thesourcecodesrepresentthefollowingcompanies:1—Mr.Fothergill’s(SouthWindsor,NSW,Australia);2—Yates(WetherillPark,NSW,Australia);3—GreenHarvest(Maleny,QLD,
Australia);4—EdenSeed(LowerBeechmont,QLD,Australia);5—SelectOrganicSeeds(LowerBeechmont,QLD,Australia).
Plants2018,7,47 6of17
Table2.PhithickeninginductionbysaltindifferentBrassicaspeciesandcultivars.
RootLength(cm) RootLengthRatio PhiThickeningScore
Species/Subspecies CommonName Cultivar SourceCode MeasurementDay
0mM 80mM (80mM/0mM) 0mM 80mM
Canola ‘Hyola97CL’ 7 5 3.6±0.8 2.5±1.0 0.69 0 41
Canola ‘Hyola474CL’ 7 5 2.7±0.6 2.1±1.1 0.78 0 25
Brassicanapus Canola ‘Archer’ 7 5 2.5±0.8 2.7±0.8 1.08 0 68
Canola ‘Edimax’ 7 5 2.6±0.6 2.0±0.9 0.77 0 8
Canola ‘Sensation’ 7 5 4.0±2.5 2.3±1.2 0.58 0 0
Brassicarapa
subsp.pekinesis ‘Wongbok’ 4 5 5.8±1.0 4.5±0.4 0.77 4 0
subsp.pekinesis ‘Michihli’ 4 10 5.1±0.7 4.8±0.3 0.95 0 0
subsp.chinensis ‘Pakchoigreen’ 4 5 3.4±0.7 3.7±0.8 1.11 0 0
subsp.chinensis ‘Granat’ 5 6 3.8±1.5 5.2±0.4 1.36 0 0
subsp.perviridis Saladgreens ‘Komatsuna’ 4 5 4.4±0.8 4.5±0.8 1.02 0 0
var.japonica ‘Mibuna’ 4 8 3.6±1.0 3.8±1.3 1.07 0 0
subsp.narinosa Saladgreens ‘Tatsoi’ 4 8 4.7±0.9 3.8±0.8 0.80 0 0
subsp.rapa Turnip ‘Goldball’ 4 5 4.6±0.8 4.6±1.3 1.00 0 0
Mustard ‘Koreanred’ 8 6 4.0±0.6 4.3±0.6 1.08 0 0
Mustard ‘Asiangreen’ 8 6 2.0±0.3 2.7±0.8 1.35 0 0
Brassicajuncea Mustard ‘broadleaf’ 8 6 3.9±0.3 3.3±0.5 0.85 0 0
Mustard ‘gaichoy’ 8 6 4.6±0.4 2.8±0.3 0.61 0 0
Mustard ‘Largeleaf’ 9 6 1.9±0.7 2.1±0.7 1.11 0 0
Blackmustard 6 5 7.3±1.1 5.8±0.7 0.79 0 0
Brassicanigra
Englishmustard 9 6 5.5±0.5 4.9±0.5 0.89 0 0
Mustard ‘Texselgreens’ 8 6 4.2±0.6 3.7±1.0 0.88 0 0
Brassicacarinata
EthiopianCabbage 3 6 4.1±0.3 4.1±0.6 1.00 0 0
Thesourcecodesrepresentthefollowingcompaniesandlaboratories:3—GreenHarvest;4—EdenSeed;5—SelectOrganic;6—TheSeedCollection(UpperFerntreeGully,VIC,Australia);
7—DrSusanSprague,CSIROAgriculture,(Canberra,ACT,Australia);8—4SeasonsSeeds(Tenterfield,NSW,Australia);9—FairDinkumSeeds(GinGin,QLD,Australia).
Plants2018,7,47 7of17
Table3.PhithickeninginductionbysaltindifferentspeciesoftheBrassicaceaefamily.
RootLength(cm) RootLengthRatio PhiThickeningScore
Species CommonName Cultivar SourceCode MeasurementDay
0mM 80mM (80mM/0mM) 0mM 80mM
Sinapsisalba Whitemustard 1 5 2.8±0.9 2.4±1.1 0.84 102 95
Iberisamara Candytuft ‘Iceberg’ 6 8 3.2±1.7 2.2 0.67 22 46
Lobulariamaritima Alyssum ‘RosieO’Day’ 4 6 1.5±0.2 2.0±0.4 1.37 15 43
Thlaspsicauelescens Alpinepennycress 13 11 3.1±0.4 1.8±0.3 0.58 15 16
Malcolmiamaritima Stock ‘Earlygiantimperial’ 4 6 3.6±0.6 2.5±0.9 0.69 0 0
Herperismatronalis Dame’swhiterocket 10 13 2.0±0.8 2.3±0.6 1.16 0 0
Diplotaxiserucoides WhiteRocket ‘Wasabi’ 10 5 2.0±0.4 1.9±0.4 0.99 0 0
Diplotaxismuralis Wildrocket ‘Saladgreens’ 4 11 1.5±0.1 1.97 1.36 0 0
Erucasativa Rocket 4 5 2.5±0.6 2.5±0.8 1.00 0 0
Erucavesicaria Arugula 8 5 2.7±1.1 4.5±1.3 1.67 0 0
Isatistinctoria Dyerswoad 10 6 2.4±0.6 2.2±0.9 0.93 0 0
Cheiranthuscheiri Wallflower ‘Fireking’ 6 5 3.5±0.6 2.7±0.8 0.77 0 0
Matthiolaincana Virginianstock 1 8 3.4±1.3 2.9±0.3 0.86 0 0
Aubrietasp. Rockcress 11 11 1.8±0.8 0.21 0.12 0 0
Lepidiumsativum Plaincress 5 5 5.5±1.3 5.2±0.9 0.93 0 0
Cardaminehirsuta Hairybittercress 12 11 1.7±0.8 1.7±0.0 1.00 0 0
Arapidopsisthaliana EcotypeCol-0 14 14 1.9±0.7 2.8±0.7 1.47 0 0
Nasturtiumofficinale Watercress ‘Aqualargeleaf’ 3 11 1.6 0.53 0.33 0 0
Barbareaverna Americauplandcress 4 35 1.1 0.9 0.81 0 0
Crambemaritima Seakale 8 35 3.8 - - 0 -
Lunariabiennis Moneyplant 10 13 3.9±1.0 - - 0 -
Thesourcecodesrepresentthefollowingcompaniesandlaboratories:1—Mr.Fothergill’s;3—GreenHarvest;4—EdenSeed;5—SelectOrganic;6—TheSeedCollection;8—4Seasons
Seeds(Tenterfield,NSW,Australia);10—SouthernHarvest(Kingston,TAS,Australia);11—BoondieSeeds(Armidale,NSW,Australia);12—DrJohnBowman(MonashUniversity,VIC,
Australia);13—DrDamienCallahan(DeakinUniversity,VIC,Australia);14—ArabidopsisBiologicalResourceCenter.
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wmeoarkel ywleaabkellyle ldabCeallsepda rCiaanspsatrriipanw sittrhiipn wthietheinnd tohde eernmdios,dwerimthisb,o wthitsht rbuoctthu rsetsruscutrurroeusn sduirnrgouthnedhinega vtihlye
lhigeanvifiileyd lixgynliefmiedv exsysleelmsi nvetshseelcse nint rtahles tceelnet.rIanl sntoenle-.i nInd uncoend-iBn.doulecreadce Ba.c ovle‘rMacaeraa cthvo ‘nMFa1ra’trhooonts ,Fo1n’ lryoothtse,
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(cFeilglsu r(Fei2gau,rSeu 2pap, leSmupepnlteamryenMtaorvyi eMS1o)v.iHe oSw1)e. vHeor,winevtheer, pirne stehne cperoefsesanlcte, aosf trsoanltg, lay slitgronnifigelyd nliegtnwifoierdk
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O(Snueppulnemuseunatlarays pMecotviwe aSs2t)h. eOnlaed udneru-sliukael naseptwecotr kwaosf twhea lllatdhdicekr-elnikine gnseptwreosrekn tofo nwathlle thinicnkeernifnagces
opfretsheenti nonne rthceo ritnicnaelr cfealclew oafl lt,hiem imnneedri actoerltyicaadl jcaeclel nwtatoll, tihmemeneddioadteelrym aisdj(aFciegnutr eto2 tbh,ea errnodwodsheermadi)s.
T(Fhiegsuerest 2rubc, taurrreoswwshilelabde).d Tishceusses esdtruinctmuroerse wdeiltla bilei nditshceusnseexdt isne cmtioorne. detail in the next section.

Figure 2. Maximum projection confocal optical stacks of berberine-stained lignin in B. oleracea roots
Figure2.Maximumprojectionconfocalopticalstacksofberberine-stainedlignininB.oleracearoots
of cultivar ‘Marathon F1’. (a) Control root in the absence of added salt. The Casparian strip (Cs) was
ofcultivar‘MarathonF1’.(a)Controlrootintheabsenceofaddedsalt.TheCasparianstrip(Cs)was
weakly labelled while the xylem bundles (x) were strongly labelled; (b) Root grown in presence of 80
weaklylabelledwhilethexylembundles(x)werestronglylabelled;(b)Rootgrowninpresenceof
8m0Mm NMaNCal,C wl,hwichhi cshtrostnrgolnyg ilnydinudceudc etdhet hfoerfmoramtioatni oonf opfhpi hthiitchkiecnkienngins g(Φs)(.Φ B)a.rB =a r1=001 μ00mµ. m.
We used the re-slice function in ImageJ to convert the data sets of roots imaged in longitudinal
Weusedthere-slicefunctioninImageJtoconvertthedatasetsofrootsimagedinlongitudinal
view into cross-section (Figure 3). We imaged ‘Marathon F1’ and confirmed that, similar to previous
viewintocross-section(Figure3). Weimaged‘MarathonF1’andconfirmedthat,similartoprevious
studies [15,18], lignified phi thickenings were induced by salt in cells in the innermost layer of the
studies[15,18],lignifiedphithickeningswereinducedbysaltincellsintheinnermostlayerofthe
cortex. These cells were immediately outside the endodermis in which berberine staining showed a
cortex. Thesecellswereimmediatelyoutsidetheendodermisinwhichberberinestainingshowed
faint signal for the weakly lignified Casparian strip (Figure 3b). This pattern, also visible with
a faint signal for the weakly lignified Casparian strip (Figure 3b). This pattern, also visible with
pontamine-labelled cellulose, represents Type I phi thickening [9,10]. The pontamine counterstaining
pontamine-labelledcellulose,representsTypeIphithickening[9,10]. Thepontaminecounterstaining
also confirmed that this layer of phi thickening-containing cells was the innermost of the three layers
alsoconfirmedthatthislayerofphithickening-containingcellswastheinnermostofthethreelayers
of cortical cells, and that these cells were smaller than the outer two (or sometime three) cortical layers
ofcorticalcells,andthatthesecellsweresmallerthantheoutertwo(orsometimethree)corticallayers
(Figure 3b). In control plants in which no induction had occurred (Figure 3a), the Casparian was again
Plants2018,7,47 9of17
(PFlaignutsr 2e031b8), .7I, nx cFoOnRt rPoElEpRla RnEtsViInEWw h ichnoinductionhadoccurred(Figure3a),theCasparianwas9a ogfa 1in7
faintlyvisible,buttherewasnoindicationofthepresenceofthephithickenings,eitherwithberberine
faintly visible, but there was no indication of the presence of the phi thickenings, either with berberine
stainingofligninorpontaminestainingofcellulose.
staining of lignin or pontamine staining of cellulose.

Figure 3. Concurrent confocal images of pontamine-stained cellulose (red in overlay) and berberine-
Figure3.Concurrentconfocalimagesofpontamine-stainedcellulose(redinoverlay)andberberine-
stained lignin (green in overlay, appearing as yellow when colocalised with pontamine-stained
stained lignin (green in overlay, appearing as yellow when colocalised with pontamine-stained
cellulose) in B. oleracea ‘Marathon F1’ roots. Images are computer-generated re-slices through optical
cellulose)inB.oleracea‘MarathonF1’roots.Imagesarecomputer-generatedre-slicesthroughoptical
stacks collected from roots mounted longitudinally. (a) Control root showing absence of phi
stackscollectedfromrootsmountedlongitudinally.(a)Controlrootshowingabsenceofphithickenings;
thickenings; (b) Root grown for 4 d in the presence of 80 mM NaCl which induced formation of phi
(b)Rootgrownfor4dinthepresenceof80mMNaClwhichinducedformationofphithickenings
(tΦhi)ckinenthinegisn n(Φer) mino tshtec ionrnteexrmimomst ecdoriatetexl yimomutesdidiaetethlye oeuntdsoiddee trhmei se.nIdnobdoetrhmrios.o Itns, bCoathsp raoroiatsn, Cstarsippsar(Ciasn)
strips (Cs) in the endodermis are weakly labelled with berberine while xylem (x) strongly labelled in
intheendodermisareweaklylabelledwithberberinewhilexylem(x)stronglylabelledinthecentral
the central vascular tissue. Bar = 100 μm.
vasculartissue.Bar=100µm.
Similar phi thickening induction was observed in 4 d-old seedlings of B. napus in a spring canola
Similar phi thickening induction was observed in 4 d-old seedlings of B. napus in a spring
cultivar, ‘Hyola971CL’, when grown on nutrient agar plates containing salt. In ImageJ-derived cross-
canolacultivar,‘Hyola971CL’,whengrownonnutrientagarplatescontainingsalt. InImageJ-derived
sections, phi thickening development was absent in control roots but induced in the innermost of
cross-sections,phithickeningdevelopmentwasabsentincontrolrootsbutinducedintheinnermost
three cortical cell layers in patterns similar to that seen in B. oleracea (Figure S1).
ofthreecorticalcelllayersinpatternssimilartothatseeninB.oleracea(FigureS1).
Several unusual aspects of phi thickening development were observed by confocal microscopy.
Severalunusualaspectsofphithickeningdevelopmentwereobservedbyconfocalmicroscopy.
The most notable of these was the development of regular, lignified thickenings on the inner face of
Themostnotableofthesewasthedevelopmentofregular,lignifiedthickeningsontheinnerfaceof
the inner cortical cells along the wall immediately adjacent to the endodermis, although variation in
theinnercorticalcellsalongthewallimmediatelyadjacenttotheendodermis,althoughvariationin
the architecture of these structures were observed across the Brassicaceae. In B. napus ‘Hyda971CL’,
thearchitectureofthesestructureswereobservedacrosstheBrassicaceae. InB.napus‘Hyda971CL’,
a network of delicate ladder-like thickenings were observed that were organised at non-transverse
anetworkofdelicateladder-likethickeningswereobservedthatwereorganisedatnon-transverse
angles (Figure 4a). In Sinapsis alba (white mustard), however, the angle of these structures was slightly
angles(Figure4a). InSinapsisalba(whitemustard),however,theangleofthesestructureswasslightly
different, thus forming a more uniform net-like structure which was extensively lignified (Figure 4b).
different,thusformingamoreuniformnet-likestructurewhichwasextensivelylignified(Figure4b).
The net-like structures in ‘Marathon F1’ and ‘Red cabbage ruby ball F1’ (data not shown) were similar
Thenet-likestructuresin‘MarathonF1’and‘RedcabbagerubyballF1’(datanotshown)weresimilar
to networks seen in S. alba (white mustard). In cross-section, these delicate wall thickenings were
tonetworksseeninS.alba(whitemustard).Incross-section,thesedelicatewallthickeningswerevisible
visible as small, lignified specks on the inner-face of the inner cortical cells (Figure 4d,e). Distinct from
assmall,lignifiedspecksontheinner-faceoftheinnercorticalcells(Figure4d,e). Distinctfromthe
the reticulate structures observed in Brassica and Sinapsis, extensions from the phi thickenings were
reticulatestructuresobservedinBrassicaandSinapsis,extensionsfromthephithickeningswereseen
seen along the cell wall between the inner cortex and the endodermis in salt-treated roots of T.
alongthecellwallbetweentheinnercortexandtheendodermisinsalt-treatedrootsofT.caerulescens.
caerulescens. Here, the lignified phi thickening extended all the way along the inner face of the inner
Here, the lignified phi thickening extended all the way along the inner face of the inner cortical
cortical cells (Figure 4f), so that in cross-section these structures appeared more like “crescent
cells(Figure4f),sothatincross-sectionthesestructuresappearedmorelike“crescentthickenings”,
thickenings”, as described in roots of several fruit trees [5,6], rather than typical phi thickenings. In
asdescribedinrootsofseveralfruittrees[5,6],ratherthantypicalphithickenings.Inlongitudinalview,
longitudinal view, the walls displayed the formation of pore-like structures between the inner cortex
thewallsdisplayedtheformationofpore-likestructuresbetweentheinnercortexandtheendodermis
and the endodermis (Figure 4c). Similar, lower-resolution images were previously shown for heavy
(Figure4c). Similar,lower-resolutionimageswerepreviouslyshownforheavymetal-inducedphi
metal-induced phi thickenings in T. caerulescens roots [25], and it is possible that structures in Thlaspi
thickeningsinT.caerulescensroots[25],anditispossiblethatstructuresinThlaspirootspreviously
roots previously described as “a second layer of endodermis cells” may also reflect this development
described as “a second layer of endodermis cells” may also reflect this development of the phi
of the phi thickening layer in the inner cortex [26].
thickeninglayerintheinnercortex[26].
These reticulate extensions of the phi thickening network from the radial walls of the inner
cortical cells to the inner face of the cells were first reported by Woronin in 1878 [17] and Van Tieghem
in 1887 [16]. However, these are the first actual images of their appearance in both longitudinal and
Plants 2018, 7, x FOR PEER REVIEW 10 of 17
Pclraontsss2-0s1e8c,t7i,o4n7 to be published. These structures would, however, be the “wall ingrowths” repor1t0eodf 1in7
electron micrographs of salt-treated B. oleracea roots [15].

Figure 4. Confocal imaging of fine reticulate networks associated with phi thickenings in Brassicaceae
Figure4.ConfocalimagingoffinereticulatenetworksassociatedwithphithickeningsinBrassicaceae
roots. (a–c) Average projections of longitudinal sections showing lignin stained with berberine; (d–f)
roots. (a–c) Average projections of longitudinal sections showing lignin stained with berberine;
Computer-generated re-slices through optical stacks collected from roots mounted longitudinally
(d–f)Computer-generatedre-slicesthroughopticalstackscollectedfromrootsmountedlongitudinally
showing berberine-stained lignin (green) and pontamine-stained cellulose (red); (a,d) B. napus
showing berberine-stained lignin (green) and pontamine-stained cellulose (red); (a,d) B. napus
‘Hyda971CL’ 5 day-old root treated with 80 mM NaCl induced the formation of phi thickenings in
‘Hyda971CL’ 5 day-old root treated with 80 mM NaCl induced the formation of phi thickenings
the innermost cortex and reticulate network (n) at the inner face of the innermost cortical layer
intheinnermostcortexandreticulatenetwork(n)attheinnerfaceoftheinnermostcorticallayer
adjacent to the Casparian strip-containing endodermis; (b,e) S. alba ‘White mustard’ 5 day-old root
adjacenttotheCasparianstrip-containingendodermis;(b,e)S.alba‘Whitemustard’5day-oldroot
treated with 1% sucrose induced the formation of phi thickenings in the innermost cortex and
treatedwith1%sucroseinducedtheformationofphithickeningsintheinnermostcortexandreticulate
reticulate network (n) at the inner face adjacent to the endodermis; (c,f) T. cauelescens 8 day-old root
network(n)attheinnerfaceadjacenttotheendodermis; (c,f)T.cauelescens8day-oldroottreated
treated with 1% sucrose induced the formation of lignified thickenings along the inner face of the
with1%sucroseinducedtheformationoflignifiedthickeningsalongtheinnerfaceoftheinnercortex
inner cortex adjacent to the endodermis. All bars = 50 μm.
adjacenttotheendodermis.Allbars=50µm.
2.4. Phi Thickenings in Brassica Oleracea Adventitious Aerial Roots
Thesereticulateextensionsofthephithickeningnetworkfromtheradialwallsoftheinnercortical
cells Etoxttehnesiinven efrorfamcaetioofnt hoef cpehllis twhiecrkeenfiirnstgsr ewpoarst eiddebnytifWieodr oinn inaeirnia1l 8r7o8ot[s1 7d]eavnedloVpainngT fireogmhe msteimn
1n8o8d7e[s1 6o]f. BH. oowlereavceear, cthve ‘sMeaarraeththoen fiFr1st’ a(Fcitguuarlei m5)a.g Tesheosfet hroeiortsa pwpeeraer arnelcaetiivnelbyo tshholortn gaintdu dcilnuasltearnedd
ctroogsest-hseerc taionnd,t uonbleikpeu pbrliimshaerdy. rTohoetss,e hsatdru ocntulyre tswwo olauylder,sh oofw ceovretirc,able cethlles.“ Pwhai ltlhiincgkreonwintghss ”wreerpeo prrteedseinnt
einle cthtreo innmneircmroogsrta cpohrstiocafls caeltl-lt lraeyaeterd asB t.hoilcerka bceaanrdoso tthsa[1t 5s]e.parated cells from one another (Figure 5a,b).
A heavily-lignified reticulate network of thickenings with irregular patterning also formed at the
2in.4n.ePrh fiaTchei cokfe nthineg isninneBr rcaossritceax OaldejraacceeantA tdov etnhtei teionudsoAdeerrimaliRs ocootsntaining Casparian strip (Figure 5b,c).
This network could also project outwards as hook-like structures from the radial bands of the phi
Extensiveformationofphithickeningswasidentifiedinaerialrootsdevelopingfromstemnodes
thickenings towards the outer wall of the inner cortical layer (Figure 5b,c).
ofB.oleraceacv‘MarathonF1’(Figure5). Theserootswererelativelyshortandclusteredtogether
and, unlike primary roots, had only two layers of cortical cells. Phi thickenings were present in