apical wall growth in fungi

14 Pages·2012·3.37 MB·English

ActaBot.Neerl.37(1), March1988,p.3-16
A steady-state model for apical wall growth in fungi
J.G.H. Wessels
DepartmentofPlantPhysiology, BiologicalCentre, University ofGroningen,9751, NNHaren,
TheNetherlands
Introduction 3
Generalperspective 3
Structureoffungal walls 6
Biosynthesis andassembly ofthewall 9
Mechanismofapical wallgrowth 11
Conclusions 13
INTRODUCTION
The presenceofcell wallsmakesplants immobilebutfungi (defined as a separate king-
dom,Whittaker1969) haveevolvedas organisms which,despitethepresenceofcellwalls,
havea certaindegreeofmobilityduetoapicalgrowth. Themycelialcolony, consisting ofa
system ofbranchedhyphae, maythus growover andthrough substrates.Consequently,
theactively growingpartofthe fungal mycelium constantly moves away fromitsoriginal
position whilecolonizing deadsubstrata(saprotrophs) or livingorganisms (biothrophs)
as inparasitic andsymbiotic associations.
Theseactivities are particularly prominent in relationto plants, as evidenced by the
fact thatfungi arethe maindecomposers ofthe lignocellulosic plant cell wall(Crawford
1981),they are also the mainplant pathogens (Dickinson &Lucas 1982)and they form
mycorrhizae withnearly allland plants(Harley & Smith 1983). Although theassociation
offungi withhealthy animals(including man) is limited, theircolonizing ability is now
becoming aproblem inimmuno-compromised patients(Chandler 1986).
Thispaperisconcernedwiththemechanisms responsible for thecontinuousextension
ofthewallattheapexofgrowing fungal hyphae. Itshouldbenoted,however,thatsome
cellsin plantsshowintrusivegrowth byapical extension,e.g. roothairs,pollen tubes, and
rhizoids(Schnepf 1986). It is only in fungi that a view ofapical wall extension can be
formulatedbasedontheknowledge oftheemergingwallstructure.Itispossible, however,
thattheprinciples involvedmayultimatelyprovetobesimilar inapically growing cellsof
both plants and fungi although detailswouldbe differentbecauseof differencesin the
chemistry ofwallpolymers.
GENERAL PERSPECTIVE
Many ofthebasicobservationsonapical wallgrowth weremadeinthenineteenthcentury
and various hypotheses thenput forward to explain apical growth still survive. Most
notableis apublication from 1892by Reinhardtinwhich hedescribedexperiments with
the wide hyphae of Peziza species showing that disturbances of growth were first
manifestedat theapices ofhyphae. Many ofthese observations were laterrepeated and
Key-words:apicalgrowth,β-glucanc,hitin,fungalcellwall,fungalhypha,Schizophyllumcommune,wall growth.
3
4 J.G.H. WESSELS
Fig.1.The rateofexpansionofanypointattheapexisproportionaltothecosineoftheangleawhentheshapeof
thetipishemispherical(a),ortotheco-tangentoftheangleawhen theshapeofthetipishalfellipsoid(b).
extendedby Robertson(1958 1965). Achange intheshape ofthe apexfrommoreor less
hemispherical tohalf-ellipsoidsofrevolutionwithincreasinggrowth rate(Fig. 1),as noted
byTrinci&Saunders(1977), wasalsoobservedbyReinhardt.Inaddition, henotedthatthe
origin ofcurvatureof hyphae was at the apexand together these observationsprovided
theevidencethat hyphal extensionoccurs at theapex. Directobservationsby Reinhardt
(1892)on the displacement ofparticleson thesurface during apical growth couldonly be
madewith root hairs ofLepidium sativum. Similarexperiments with a fungal system
(Castle 1958) confirmedthevalidity ofhis conclusionthat fungal hyphae extend attheir
apices.
Withregard to themechanisms involved inapical wall growth, Reinhardt(1892) dis-
cussed thethen prevailing theory of leading botanists who regarded enlargement ofthe
wall areaas a process in which anelasticor plastic wall expands due to turgorpressure
while new wall materialis added by apposition or intussusception. However, he con-
sidered such a theory inadequate because it would require an increase in mechanical
strengthofthewallfromtheveryapextothebaseoftheextensionzone.Asheputsit, such
anincreaseinstrength couldbeachievedby aproportional increaseinwallthicknessorby
achange in thequalityofthemoleculesthatmakeupthewall. Hefoundnoevidencefora
change in wall thickness. Later investigators (Girbardt 1969, Grove & Bracker 1970,
Trinci &Collinge 1975) also foundthatwallthickness remains uniformin theextension
zone. Achange inthequalityofthewall molecules—which ispreciselythekindofchange
which is suggested by recent work from our laboratory (see below)—was considered
unlikelyby Reinhardt.Thusonewouldexpect thatanincreaseinhydrostatic pressure,as
caused by flooding with water, wouldresultin swelling or bursting at the extreme tip.
Insteadheobservedthatbursting occurredjustundertheapexwherethecylindrical form
is attainedandwhere thecircumferentialstressin thewall increases.Subapical swelling
and bursting was also observedby laterinvestigators (Robertson 1958, 1965,Bartnicki-
Garcia&Lippman 1972,seeFig. 2).Reinhardtconcludedthatthewallmusthaveuniform
strength over the whole apex and does not grow by plastic expansion. He proposed
intussusception ofwallmaterialmaximally attheextremetipand declining tozero atthe
baseoftheextensionzone. Thefactthatreliefofturgorpressure,by applying solutionsof
lowosmoticpotential, resultedincessationofgrowth attheapexwasinterpreted as being
due to detachmentofcytoplasm from the apical wall, thus interrupting the organized
delivery ofnewwall materialbythecytoplasm.
Disregarding theobservations madeby Reinhardt(1892), and probably inspired by
considerations ofD’Arcy Thompson (1917, see Bonner 1961) on the origin of cellular
form, mathematicalmodels have been put forward to describe apical morphogenesis.
APICALWALL GROWTHIN FUNGI 5
Fig. 2.Explosive apicalbursting ofagar-grown hyphaeofSchizophyllumcommuneafterfloodingwith 0-5%
acetic acid, followingtheprocedureofPark & Robinson (1966).The cytoplasm is extruded throughahole
locatedatthebaseoftheapicaldome(a)orthewholeapicaldomeisblown off(b).(CourtesyofDrJ.H.Sietsma.)
They allrely on theconceptthatextensionatthe hyphal apexis due to thepresence ofa
gradientintheplasticity ofthewallsuch thatthereisa decreaseinthetendency ofthewall
to yield to turgorpressure fromtheextreme tip downwards(de Wolff& Houwink 1954,
D-1 RivaRicci& Kendrick 1972,Green 1974,Trinci& Saunders1977,Saunders& Trinci
1979, Koch 1982). In all these models expansion at any point on the apical domeis
determinedby its position, according to amathematicalfunctionwhich depends onthe
shape oftheapex(Fig. 1).To putthese modelsthrough atestitwouldseemnecessaryto
determinethe plastic andelasticpropertiesofthewallatvariouspointsover thegrowing
apex;obviously such measurements are difficultto make.
IfReinhardt’s theory is to be dismissed, another explanation must be foundfor the
tendency ofhyphal tips to swelland burst atthe baseofthe apical domeinsteadofthe
extremeapexwhensubjected tohigh turgorpressure.Itis unfortunatethatnoneofthose
who haveadvancedatheory foraplastic wallexpanding underturgorpressure has cared
to explainsubapical bursting.Possibly therecently discoveredcytoskeletal elementsinthe
hyphal apex(forreview seeMcKerracher& Heath 1987) protect thenewly formed deli-
cate wallfrombecoming subjectto high hydrostaticpressure(Wessels 1986).Thishasalso
beenproposed fortheapical wallinextending pollen tubes(Picton & Steer 1982).Protec-
tion ofthe apical wall against high internalpressure by the underlying structuredcyto-
plasm mayexplain why thewalldoesnot alwaysburstatitsweakestpoint, i.e. atthevery
apex,when turgorissuddenly increased.
Atthesametime,recognitionofthepresenceofcytoskeletal elementsattheapexwould
question thevalidityofmathematicalmodelsassuming expansion underturgorpressure.
Such models may be wrong in assuming thatturgor pressure, probably generated sub-
apically in vacuoles, is uniformly present over the whole apical wall area. New models
6 J. G.H. WESSELS
taking into account the presence of cytoskeletal elements might assign a role to the
cytoplasm inshaping thehyphal apex(asalready advocatedbyReinhardt)whileretaining
theconceptof a deformablewall expanding underthe influenceof hydrostatic pressure
generated insubapical hyphal parts.
Ifthereisagradientintheplasticity ofthewall,maximalattheveryapexanddeclining
towardsthe baseoftheapical extensionzone,thenhow is this gradient generated? Two
possibilities were consideredinthe nineteenthcentury. Eitherthe wallisoriginally plastic
andexpands untilit becomesrigid or thewall is synthesized as a rigid entity andcannot
expand untilitbecomesplastic. Thefirstconceptwasgenerally accepted inthenineteenth
century; controversies mainly concerned whetherwall additionwas by intussusception
(Nageli) orapposition (Strasburger) (cit: Reinhardt 1892). Thisconceptofrigidification
ofthewallafteritsformationwas implicit in formulationsby Robertson(1958, 1965) to
explain his observations on living hyphae. Work on wall biogenesis in Schizophyllum
commune(to be discussed below) has given this conceptamolecularand experimental
basis(Wessels &Sietsma 1981b,Wessels etal. 1893)andhasbeennamedthe‘steady-state
modelofapicalwallgrowth’(Wessels 1986).Thealternativeconceptthatthewallmustbe
continuously loosenedbylyticenzymesinordertoexpand wasexpressed acenturyagoby
Marchall-Ward (1888)and has morerecently beenfavouredby Bartnicki-Garcia (1973)
andGooday(1978). Although directevidenceforthisconcept,whichpresumesa‘delicate
balance between wall synthesis and wall lysis’ (Bartnicki-Garcia 1973),is missing, it is
rather uncritically cited as afact inmanyresearchpapersandtextbooks.Ihavecritically
examinedtheevidenceandhavecometo theconclusionthat,todate,theevidenceforthis
concept is very thin indeed (Wessels 1984, 1986). Lysins play no role in the preferred
steady-state modelofapical wallgrowth. However,thismodeldoesnotdisprove arole for
lyticenzymes.Forthemoment itseemssufficientto explainthestructuralandexperimen-
tal data. Ifhard evidence for a role of lysins in apical wall growth were to arise, e.g.
through genetic studies, thiswouldnecessitatemodificationofthemodel.
STRUCTURE OF FUNGAL WALLS
At first sight fungal walls containa bewildering number ofpolymers and constituent
monomers (forreviews seeBartnicki-Garcia 1968, Wessels & Sietsma 1981a, Bartnicki-
Garcia & Lippman 1982, Wessels 1986).However, whenonly the alkali-insolublecom-
ponents are considereda much more simple picture emerges. Thealkali-insolublewall
portions ofascomycetes and basidiomycetes mainly consistof(1->4)-P-D-glucosamino-
glycans, [poly-A-acelylglucosamine (chitin) and partially orwholly deacetylated deriva-
tives (e.g. chitosan)] and (l-»3)-p-D/(l->6)-P-D-glucan. There is evidence that this
alkali-insoluble portion of the wall is solely responsible for hyphal morphogenesis
(Sietsma & Wessels 1988) and for the sake ofthe present discussionthe alkali-soluble
componentsofthewallwill thereforebelargely ignored.
In order to reveal the molecular architecture ofthe hyphal wall, electronmicroscope
observations, combinedwiththe useofmoreorless specific enzymic orchemicalextrac-
tions, have been made on the walls ofa varietyof fungi (lit.cit. in Wessels & Sietsma
1981a). On the basisofsuch observations, Hunsley & Burnett(1970) havemodelledthe
wall as aco-axially layered structure. As Burnett(1979) has pointed out, it should be
understoodthattheco-axiallyarrangedregions are notsupposed tobe discretebutgrade
intoeachother.Wessels&Sietsma(1981a), however, notedthatthe techniques usedcan
APICALWALL GROWTH IN FUNGI 7
Fig. 3.Model ofthe maturehyphalwall ofSchizophyllumcommune.Partially crystallizedchitin chains(a)are
hydrogen-bondedtochitin chains whichcarry covalentlylinked p-glucan chains.The couplingfragment(b)
contains aminoacidswith ahighproportionoflysine. The P-glucanchainsare(I->3)-Plinked andcarrysingle
(1-*6)-P-linkedglucosebranches(c)orlonger(I->6)-p-linkedglucanbranches(d)oralternatively,(1-»3)-Pand
(1-*6)-Plinked glucanbranches (e).Some unsubstituted orsparselybranched (l->3)-P glucansegmentsmay
formtriplehelices(h)whichaddtothestrengthoftheglucannetwork. Crystalline(1-*3)-a-glucanfibrils(alkali-
soluble s-glucan)(f)occurthroughoutthewall andaccumulateattheoutersurfaceasalayer.Free water-soluble
(1->3)-P-glucanchainswith single(1->6)-P-linked glucosebranches (g)arealsopresentin thewall and maybe
excreted into themedium.(AdaptedfromWessels&Sietsma1981b.)
easilyleadto misinterpretationsofwallstructure. Theyconsideredmostpublished studies
tobeinagreementwithamodelofthewallinwhichthevariouswallcomponentsare more
closely associated witheach otherto formessentially onelayer, withsome components
accumulating at the outside apparently forming extra layers. This simple modelonly
applies tovegetative hyphae andnotto thewallsofspecialized structures,e.g.sporesand
aerial hyphae, wheregenuine outerlayers maybepresent.
Figure3 depictsamodelofthehyphalwallofthebasidiomycete Schizopyllum commune
integrating the resultsof a number ofchemical, enzymic and ultrastructural analyses
(Wesselsetal. 1972,Sietsma& Wessels 1977,1979,vanderValketal. 1977).Inthiscase a
water-solublegel-like(1->3)-(3-/(l-»6)-P-glucan andawater-insolublebutalkali-soluble
(1 ->3)-ct-glucan(s-glucan) accumulateattheoutsideofalayer whichcontainsanalkali-
insoluble glucosaminoglycan-glucan complex. In this complex the glucan chains are
(1 -»3)-P-linked with(1 ->6)-p-linked branchesattached. Insomeofthechains, branches
consist ofjust one glucose residue and these chains thus resemble the gel-like glucan
accumulating on the outside ofthe hyphae. Other(1->3)-P-linked chains carry longer
(I->6)-P-linked glucan branches. Both types of branched glucans are thought to be
attached to (l-»4)-P-linked glucosaminoglycan chains through theirreducing ends via
8 J.G.H. WESSELS
Table 1.Solubilizationofnon-aminoglycansbydepolymerizationofaminoglycans*
NNiittrroouussaacciiddttrreeaattmmeenntt CChhiittiinnaasseettrreeaattmmeenntt
AAmmiinnooggllyyccaann NNoonn--aammiinnooggllyyccaann AAmmiinnooggllyyccaann NNoonn--aammiinnooggllyyccaann
ddeeggrraaddeedd rreelleeaasseedd ddeeggrraaddeedd rreelleeaasseedd
SS..ccoommmmuunnee ——((110000))tt 1100((9955-44))tf 9988 8888
AA..bhiissppoorruuss
VVeeggeettaattiivveemmyycceelliiuumm 4411 3388 6677 4488
FFrruuiitt--bbooddyyssttiippee 1199 4411 9944 5566
MM..mmuucceeddoo 7799 110000 — —
*In S. communeand A.bisporus figuresrefer tothepercentagesofaminoglycan and glucan in the alkali-
insoluble wall fractionbecomingsoluble inwaterand alkaliafterthetreatments.InM.mucedo thefiguresrefer
to the percentages ofaminoglycan and glycuronanin the whole wall whichwere soluble in water after the
treatment.Nitrousacid treatments weredone beforeexposure ofthe wallsto alkali. Chitinase treatmentswere
doneonalkali-insoluble wall fractions.
tNitrous acidtreatmentafterheatingthewallfractionin 10MNaOH todeacetylatechitin.
aminoacids, particularlylysine. Probably thesesubstituted glucosaminoglycan chainsare
partially hydrogen-bonded tomicrofibrillarchitinconsisting ofhydrogen-bonded unsub-
stituted chains ofpoly-TV-acetylglucosamine. The presence of hydrogen-bonded triple
helicesamongthe glucan chainswas inferredfromtheweak hydroglucan reflectionsseen
inX-rayanalysis ofthechitin glucan complex.Treatmentofthecomplex withhotdilute
acidbreaksthelinkages betweenglucosaminoglycan andglucan andhydrolyses (1->6)-P-
linkedglucan chainsthusleading tosharp X-rayreflectionsofhydroglucans andchitin.
The most important evidence for postulating linkage between glucan and glucos-
aminoglycan chains is that the glucan chains become soluble in water or alkali after
specific depolymerizationof(acetyl)glucosamine-containing polymers (Stagg &Feather
1973,Sietsma& Wessels 1979).Such depolymerizations (employing nitrousacid tobreak
bonds in the glucosaminoglycans where non-acetylated glucosamine residues occur
(Datema et al. 1977b) and chitinase to break bonds in homopolymer stretches of
jV-acetylglucosamine) have been shown to solubilizeall the alkali-insoluble P-glucans
fromthewall ofa numberofbasidiomycetes and ascomycetes (Sietsma & Wessels 1981;
Mol et al. 1988). Recently we foundthat even the very small amount of glucosamino-
glycan in thewall ofSaccharomyces cerevisiaemustbe heldresponsible for keeping the
P-glucan in thewallin analkali-insoluble form(Mol &Wessels 1987).
Table 1 shows data for two basidiomycetes, Schizophyllum commune (Sietsma &
Wessels 1979) and Agaricus bisporus (Mol & Wessels unpublished). In the first species
nearly all theglucosaminoglycan is acetylated andnitrousacid has littleeffectunlessthe
glucosaminoglycan is first deacetylated. Chitinase(from Serratia marcescens), on the
otherhand, is veryeffectivein degrading thisacetylated glucosaminoglycan (chitin)and
solubilizing the P-glucan. In Agaricus bisporus however, a large numberofglucosamine
,
residuesin the glucosaminoglycan occur ina deacetylated formbecause thepolymer is
extensively degraded by nitrousacidreleasing P-glucan intosolution.Consequently, the
glucan isless effectively released bychitinasebuta nitrousacid treatment,followedby a
chitinasetreatment,effectively brings alltheP-glucan intosolution(Mol&Wessels 1988).
Table 1 also shows dataderivedfromastudy onthewall ofMucormucedo(Datema etal.
1977b). In this zygomycete an even larger fractionofthe glucosamine residues of the
APICALWALL GROWTH IN FUNGI 9
Fig.4.Possible interactionsbetween glucosaminoglycans( ) and (I-*3)-p-glucan(~)involving hydrogen
bondingbetween heterologouschains.
glucosaminoglycans is deacetylated resulting in apoly-cation. Depolymerization ofthis
glucosaminoglycan releases an anionic heteroglucuronan containing fucose, mannose,
galactose and glucuronic acid (5:1:1:6 on amolecularbasis). Since in this case thenon-
aminoglycan canalsobeextractedbystrongsaltsolutionsandpartly byalkali, thelinkage
betweenthetwo polymers is probably ionicandnot covalent, as surmisedintheascomy-
cetesandbasidiomycetes.
Thelinkage ofthe 0-glucan chainstoglucosaminoglycan chainsinthewallsofascoco-
mycetes andbasidiomycetes not onlyleadstoinsolubilizationoftheglucan chainsbut, in
combinationwith hydrogen-bonding between homologous chains, mayalso resultin a
highlycross-linkedcomplex as showninFig. 3.Thismay,however, representanextreme
case.
Figure 4schematically depictssomewall structures whichmayactually arisefromthe
interactions between glucosaminoglycan and (1 ->3)-p-glucan chains. Included are
possible interactions between homologous chains involving hydrogen bondsleading to
theformationofchitinmicrofibrilsinthecaseofacetylated glucosaminoglycans andtriple
helices in the case of(l->3)-P-glucans (Jelsma & Kreger 1975). Figure 4e thus shows a
situationin whichallthechainsare involvedinhydrogen bonding whiletheouterchains
ofthe chitin microfibrilscovalently bind the glucan chains. In fact, all the structures
depicted (Fig. 4a-e) may actuallybe presentinthewalland contributeto itsmechanical
properties.
BIOSYNTHESIS AND ASSEMBLY OF THEWALL
Mostevidence (Vermeulen etal. 1979and referencescitedtherein) indicatesthat chitin
chains are made atthe outer surface ofthe plasma membraneprobably by an integral
membraneprotein, chitinsynthase, that accepts the precursor
acetylglucosamine atthe cytoplasmic sideofthe membrane.Chitosomes(Bracker etal.
1976)are minivesicleswhichprobably actas conveyersforinactivechitinsynthase enroute
10 J. G.H. WESSELS
to theplasma membrane, although the discoverersofthese chitosomes stillentertainthe
possibility thatchitosomes themselvessynthesize chitinaftertheirextrusionthrough the
plasma membraneinto thedomainofthe wall(Bartnicki-Garcia & Bracker 1984, Ruiz-
Herrera 1984).In Saccharomyces cerevisiaeevidence for vectorial synthesis ofchitin by
plasma membraneshas comefromCabib’slaboratory (Cabib’s et al. 1984) butcompli-
cations have arisen after the genefor this chitin synthase had been cloned, inactivated
invitrobyaninsertion,andusedtoreplace thewild-typeallelebytransformation(Bulawa
etal. 1986).Thegenereplacement abolishedtheappearanceofthechitin synthase studied
thus far in vitro but didnot affect synthesis of chitinin vivo, apparently catalysed by
anotherenzyme(Orlean 1987).However, autoradiography after labelling S. communein
vivowith tritiated-JV-acetylglucosamine has shownthat akali-insolubleglucosaminogly-
can is indeedsynthesized just outside the plasma membrane(van der Valk & Wessels
1977).Inthiscase theultimateproductwas mainlypoly-A'-acetylglucosaminc (chitin),but
in other cases the product may be subject to enzymic deacetylation immediately after
synthesis(Davis & Bartnicki-Garcia 1984).
Numerous studies have demonstrated the synthesis of an alkali-soluble(1->3)-(3-d-
glucan from uridine-diphosphate-glucose by membranouspreparations from fungi (lit.
cit: in Sonnenberg et al. 1985; Szaniszlo et al. 1985) but only in S. cerevisiae has the
glucan synthase rigorously been shown to be (partially) associated with the plasma
membrane(Shematek etal. 1980).
Since the polymerization of the W-acetylglucosamine residues at the outer plasma
membranesurfaceimmediately results in awater-insolubleandalkali-insolubleproduct
(Wessels et al. 1983), it followsthatafter synthesis ofthe individualglucosaminoglycan
and (3-glucan chains (indicated in Fig. 4a) the processes that lead to cross-linked wall
structures (indicated in Fig. 4b-e) must all take place within the domainofthe wall.
Becausethenature ofthecoupling fragment between theglucosaminoglycan andglucan
chainsis insufficiently known, and may differamong fungi, themechanismofcoupling
remainselusive. In addition, because ofthe polymeric nature ofthe tworeacting com-
ponentsitisunlikely thatthecoupling itselfismediatedbyanenzyme.Ratherweenvisage
aschemein whichaminoacidssuch aslysine orphenolic residuesareattachedtoamino-
sugarswithintheglucosaminoglycans and/ortothereducing endsofP-glucans. Coupling
mightthen occur viaradicals produced by oxidases in the wall. As an analogy wemay
recallthecross-linkingbetweenlysine residuesincollagen as occurringintheextracellular
matrixofanimals(Hay 1981) or thecross-linking oftryptophane residuesin extensin as
occurring in cell walls of vascular plants (Wilson & Fry 1986). Alternatively, lysine
residuesattached to aminosugars withinthe glucosaminoglycans may directlyandnon-
enzymically interact withthe reducing end of glucan chains forming a Schiff baseand
Amadoriproducts (Monnier etal. 1984).Also directinteractionsofthereducing endsof
glucan chainswithamino-groups ontheglucosaminoglycans cannotbeexcluded.
Irrespective ofthe typeofcross-links between the glucosaminoglycans andP-glucans,
post-synthetic transitionswithinthewall, suchas depictedin Fig. 4,mayhaveimportant
biological consequences. Forinstance, itcanbe envisaged that the highly hydrated pre-
cursor wall structure shown in Fig. 4ahas visco-elastic properties andcan bedeformed;
whereasthecross-linkedcomposite structuresshowninFig. 4c-edisplayvarious degrees
ofrigidity.
TheevidencecollectedwithS. communetosupportsucha two-stepprocessofsynthesis
andassembly ofwallcomponentswithanaccompanying change inmechanicalproperties
can besummarizedas follows.
APICAL WALLGROWTH INFUNGI 11
1. Water-solubleand alkali-soluble(l-»3)-(3-glucans are the precursor moleculesfor
thealkali-insoluble P-glucans inthewall.Thiswasshownbypulse-chase ofradioactivity
both with regenerating protoplasts (Sonnenberg et al. 1982) and with growing hyphae
(Wessels etal. 1983).
2. Inthepresenceofpolyoxin D, protoplasts can formawallmadeof(I->3)-a-glucan
without chitin and alkali-insoluble glucan (de Vries & Wessels 1975, van der Valk &
Wessels 1976). Although the soluble(l->3)-(3-glucan precursor moleculesare normally
formed they can then not be linked to an alkali-insolubleglucosaminoglycan and thus
remainsoluble(Sonhenberg etal. 1982).Similarly, Elorza etal. (1987) haveshown that
nikkomycin, another specific inhibitorofchitin synthase, inhibitsthe conversion ofan
alkali-soluble glucan into an alkali-insoluble glucan on regenerating protoplasts of
Candidaalbicans.
3. Autoradiography shows that alkali-insoluble glucosaminoglycans are maximally
synthesized attheextreme hyphal apexdecreasing insubapical direction.Asimilargradi-
ent is foundforwater-soluble0-glucan butvery littlealkali-insolubleglucan ispresentat
theextremeapex.Whiledisplacedinasubapical directionduringextensionintheabsence
oflabel, thewater-solubleglucans become alkali-insoluble.Ifextensiondoes not occur
duringthechase, thewater-solubleglucan canbe seentobecomeinsolubleoverthewhole
apex(Wessels etal. 1983).
4. Electron microscopy ofshadowed preparations combinedwith autoradiography
(Vermeulen & Wessels 1984) hasshown that thealkali-insoluble glucosaminoglycans at
thegrowing hyphal apexare non-fibrillarandvery susceptible tochitinaseandhotdilute
mineralacid. Thisis takenas evidencethat atthe growing apextheglucosaminoglycans
havenot yetcrystallized andare stillavailableforlinkageto glucan chains. Hyphae that
haveceasedtogrownotonly havealkali-insolubleglucan butalsomicrofibrillarchitinin
their apical walls. Recently, it was shown that also in vitro a time gapexists between
synthesis andcrystallization ofthechitin chains(Vermeulen & Wessels 1986).
5. High shearing forces, as generated by the passageof hyphae through an X-press,
disrupt growing tips and remove the pulse-labelled glucosaminoglycan and glucan.
However, after a chase ofa fewminutes these labelledpolymers, presentsubapically in
growing hyphae or apically inhyphae not growing after the chase, are not removed by
theseshearing forces(Wessels etal. 1983).Thisistheonlydirectevidencewehavetoshow
that the mixture of individualglucosaminoglycan and glucan chains present at the tip
mechanically differsfromthe cross-linkedcomplex in thematuredwall.
MECHANISM OF APICALWALL GROWTH
Theevidencesummarizedaboveserves as thebasis forthe steady-state growth modelas
shows inFig. 5. Intheextensionzone, individualglucosaminoglycan and(1->3)-P-glucan
chainsare deposited into thewall by apposition. They are probably polymerized at the
membrane-wallinterface.Per unitarea maximal deposition occurs attheextreme apex
andthen declinestowards the base ofthe extentionzonein accordancewithautoradio-
graphic estimates ofincorporation oflabelledprecursors (Gooday 1971, Wessels etal.
1983).Theglucosaminoglycan andP-glucan chainsextrudedintothewallaresupposed to
be highly hydrated and to form a visco-elastic wall that expands under the internal
pressure ofthecytoplasm. As a visco-elastic wall segment is stretched and displaced in
subapical directionby growth, it also moves to amore externalsideofthewall because
appositional additionofvisco-elasticwallmaterialontheinsidecontinuessothatuniform
12 J.G.H. WESSELS
Fig.5.Steady-statemodel ofapicalwall growth.Glucosaminoglycan( )and (I->-3)-P-glucan(~) interact
whilebeingdisplacedinasubapicaldirection. (1-*6)-p-linkedglucoseresidues also appearsubapically.When
extensionceasestheinteractionsoccuroverthewholeapex.
wallthicknessismaintained(Green 1974).Atthesametimethesubapically andexternally
displaced wall segment undergoes rigidification because of covalent bonding between
heterologous chainsand hydrogen bonding between homologous chains. At point c in
Fig. 5 theseprocesses haveadvancedtosuch anextent thatthewall nolongeryields tothe
internal pressure and the maximal diameterof the hyphae is attained. However, the
interactionsbetweenthepolymers continue(Fig. 5d) andprobably lead tofurtherrigidifi-
cation, as deduced from continued insolubilizationof0-glucan chains (Wessels et al.
1983).AlsoshowninFig.5dare(1-»6)-[3-linkedglucan chainsorglucoseresiduesappear-
ing as brancheson the(1->3)-P-linked glucan chains.Such(1->6)-(3linkages are initially
very scarce inthe insolubleglucan but rapidly appearwhenthewallmatures,eventually
outnumbering the(1 ->3)-P*linkages (Sietsma etal. 1985).It isalso possible thatthey are
part ofa mixed-linkage glucan that is secondarily hooked upto theglucosaminoglycan
chains. Itshouldalsobe stressed thatthe finalwall structure probably deviatesfrom the
highly regular structure depicted inFig, 5d. In fact,the alkali-insolublecomplex shows
very little crystallinity in X-ray diffraction and may actually be made up of various
structures as depicted in Fig. 4. Alsonot shown are the water-soluble(1-»3)-p-glucan
chainswithsingle (1 ->6)-P-linkedglucose residuesattachedwhichfreely occur inthewall
and thealkali-solubleandpartly crystalline(1 ->3)-a-glucan (s-glucan)whichaccumulates
atthe outersurfaces ofthewall.
The model described above is a steady-state model because continuedextension
depends on the continuous extrusion ofthe visco-elastic wall material which is subse-
quently hardenedby thecross-linking process inthewall. Therate ofextrusion ofwall
polymers into the wall and the rate ofcross-linking (rigidification) are supposed to be
independent. Consequently, whengrowth ceases cross-linking and stiffening will occur
over the wholeapex (Fig. 5). Theexperimental evidence doesindeedshow thatthewall
overtheapexthenassumesthesamestructureasinsubapical parts,including theappear-
ance of(l->6)-(3-linked glucose residues and chitin microfibrils (Vermeulen & Wessels
1984,Sietsmaetal. 1985).