Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms

14 Pages·2017·1.65 MB·English

Evolution of Xylan Substitution Patterns in
Gymnosperms and Angiosperms: Implications for
Xylan Interaction with Cellulose1[CC-BY]
Marta Busse-Wicher*2, An Li2, Rodrigo L. Silveira, Caroline S. Pereira, Theodora Tryfona,
Thiago C. F. Gomes, Munir S. Skaf, and Paul Dupree*
Department of Biochemistry and The Leverhulme Trust Centre for Natural Material Innovation, University of
Cambridge, Cambridge CB2 1QW, United Kingdom (M.B.-W., A.L., T.T., P.D.); Institute of Chemistry, University
of Campinas-UNICAMP, Campinas, SP 13084-862, Brazil (R.L.S, C.S.P., M.S.S.); and Department of Chemistry,
Instituto Tecnológico de Aeronáutica, Praça Marechal Eduardo Gomes, SP 12228-900, Brazil (T.C.F.G.)
ORCID IDs: 0000-0001-6290-2462 (M.B.-W.); 0000-0001-9515-3082 (R.L.S.);0000-0002-0248-0883(C.S.P.);0000-0002-1618-3521 (T.T.);
0000-0001-7485-1228(M.S.S.);0000-0001-9270-6286(P.D.).
Theinteractionbetweencelluloseandxylanisimportantfortheload-bearingsecondarycellwalloffloweringplants.Basedon
the precise, evenlyspacedpattern ofacetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recentlyproposed
that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose
microfibrils.Ingymnospermcellwalls,anyroleforxylanisunclear,andglucomannanisthoughttobetheimportantcellulose-
binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer,
Gingko,Cycad,andGnetophyta).Conifer,Gingko,andCycadxylanlacksacetylationbutismodifiedbyarabinoseandMeGlcA.
Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on
every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan,
lackingarabinosebutpossessingacetylationonalternatexylosylresidues.Alltheseprecisesubstitutionpatternsarecompatible
with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable
bindingof2-foldscrewconiferxylantothehydrophilicfaceofcellulosemicrofibrils.Moreover,thebindingofmultiplexylan
chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan
substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold
screwxylan bindshydrophilicfacesofcellulose ineudicots, early-branchingangiosperm,andgymnosperm cellwalls.
Theplantsecondarycellwallisacomplexnetworkof
1This work was supported by the Leverhulme Trust Centre for
variouspolysaccharidesandphenoliccompoundsthat
NaturalMaterialInnovation(M.B.-W.andP.D.),byTheLowCarbon
act in concert to provide strength to the cell wall
EnergyUniversityAlliance(A.L.),byGrantBB/G016240/1fromthe
BBSRCSustainable Bioenergy CentreCell WallSugars Programme (Kumar et al., 2016). Cellulose, formed of strong crys-
(T.T.andP.D.),andtheSaoPauloResearchFoundation(R.L.S.,C.S.P., talline fibrils of linear b1,4 glucan, comprises about
M.S.S.,andT.C.F.G.;Grants2013/08293-7,2014/10448-1,and2015/ 40% of dry plant biomass. The other secondary cell
25031-1). wallpolysaccharides,largelyxylanandglucomannan,
2Theseauthorscontributedequallytothearticle. comprise about 30% of dry plant biomass. The abun-
*[email protected]@cam. dance and structure of these hemicelluloses vary with
ac.uk.
plantspeciesandtissues,buttheyhaveincommonthat
Theauthorresponsiblefordistributionofmaterialsintegraltothe
theyaretightlyassociatedwithcellulose.Itisbelieved
findings presented in this article in accordancewith the policy de-
that the most important biological role of hemicellu-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
loses is their contribution to strengthening the cell
PaulDupree([email protected]).
M.B.-W. conceived the project, designed the experiments, per- wall by interaction with cellulose and, in some walls,
formedandsupervisedPACEexperiments,analyzedthedata,and withlignin(SchellerandUlvskov,2010).However,itis
wrotethearticlewithcontributionsofalltheauthors;A.L.performed unclear how hemicelluloses interact with cellulose in
mostPACEandMSexperiments,analyzedthedata,andprepared the cellwall (Cosgrove and Jarvis, 2012). In this work,
figures; T.T.performed, supervised, and analyzedMS experiments we were interested in the interaction of cellulose with
andcontributedtothewriting;R.L.S.andC.S.P.designed,preformed, xylan, one of the most abundant polysaccharides in
andanalyzedMDsimulations;T.C.F.G.designed,performed,andan-
nature.
alyzeddockingexperiments;C.S.P.,R.L.S.,andT.C.F.G.contributedto
Tounderstandpolysaccharideinteractionsinthecell
thewriting;M.S.S.supervisedandanalyzedMDexperimentsandcon-
wall, we need to know not only the hemicellulose pri-
tributedtothewriting;P.D.conceivedtheproject,designedandsuper-
mary structure, but also the conformation of the poly-
visedtheexperiments,analyzedthedata,andwrotethearticle.
[CC-BY]ArticlefreeviaCreativeCommonsCC-BY4.0license. saccharide chains. The glucan chains in cellulose are
www.plantphysiol.org/cgi/doi/10.1104/pp.16.00539 similartoaflatribbonknownasa2-foldhelicalscrew
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Interaction of Xylan with Cellulose in Gymnosperms
and associate through lateral hydrogen bonding into bearssubstitutionssolelyonalternatexylosylresidues.
sheets.Thesheetsofglucanchainsstackontopofeach Every second Xyl is acetylated (Busse-Wicher et al.,
other, resulting in highly ordered crystalline cellulose. 2014; Chong et al., 2014), and MeGlcA side chains re-
It is still unclear how many glucan chains form a mi- sideonevenlyspacedxylosylresidues,largelyat6-,8-,
crofibrilandwhetherthemicrofibrilhasahexagonalor 10-,or12-residueintervals(Bromleyetal.,2013).Inthis
rectangularcrosssection.However,recentstudiesshed scenario,onaxylanbackboneintheribbon-like2-fold
somelightontothesequestions(Fernandesetal.,2011; helical screw conformation, all the decorations will
Newman et al., 2013; Thomas et al., 2013; Cosgrove, face one side, creating an unsubstituted xylan surface.
2014;Oehmeetal.,2015;Thomasetal.,2015;Wangand Therefore,in additionto formingstacking interactions
Hong,2016;Vandavasietal.,2016).Whichevermodelis on the hydrophobic surface, this xylan structure is
favored,hydrophobic(e.g.100or200)andhydrophilic compatible with hydrogen bonding to the hydrophilic
(e.g.110or010)crystalfacesareexposedforinteraction surface of cellulose (Busse-Wicher et al., 2014; Busse-
with other molecules such as hemicelluloses (Zhao Wicheretal.,2016).
etal.,2014;Cosgrove,2014;Lietal.,2015). Both xylan and glucomannan are substantial com-
Theb1,4xylanbackboneisalwaysfurthermodified, ponentsofvascularplantsecondarycellwalls(Timell,
oftenbyacetyl(Ac),arabinosyl(Ara),andglucuronosyl 1967; Willfor et al., 2005; McKee et al., 2016). In
(MeGlcA)side-chainsubstitutions.Thesesubstitutions conifers (gymnosperms, Pinales), the main hemicellu-
are supposed to be necessary to maintain xylan solu- lose is glucomannan, but eudicots possess relatively
bility (Mikkelsen et al., 2015). Unsubstituted xylan littleglucomannan(SchellerandUlvskov,2010;Huang
forms crystalline fibers of chains adopting a 3-fold etal.,2015),andthesecondarycellwallsaredominated
screw helix (Nieduszynski and Marchessault, 1971). by xylan, suggesting xylan might have adopted addi-
Consequently, xylan substitutions are essential for tionalfunctionsinthesefloweringplantsthatproduce
xylan function and vascular plant viability (Mortimer hardwoods (Dammström et al., 2009). Conifers, pro-
et al., 2010; Xiong et al., 2013, 2015). In vitro experi- viding softwood for the paper, pulp, and construction
ments and in silico modeling suggest xylan interacts industries, are of major ecological and economical
with cellulose, and it is widely accepted that this is value. Consequently, understanding the function and
partlythroughinteractionsonthehydrophobicfacesof architecture of the cell wall components of softwoods
thecellulosefibrils(Bosmansetal.,2014;Köhnkeetal., andhardwoodsisofgreatimportance.
2011; Kabel et al., 2007; Busse-Wicher et al., 2014). In To investigate whether the precise arrangement of
contrast to the binding to the hydrophobic faces, the xylandecorationsonevenlyspacedxylosylresidues,as
backbones of highly substituted hemicelluloses are seenineudicots,isanovelfeatureofhardwoodxylan,
thoughttobeunabletohydrogenbondeffectivelywith we analyzed the pattern of xylan substitution in vari-
the hydrophilic surfaces of cellulose fibrils because of ous gymnosperms and angiosperms. In addition to
sterichindrance.Forexample,hydrogenbondingofthe conifers, there are threefurther gymnosperm lineages:
xyloglucanbackbonetocellulosewouldbeblockedby Cycad,GingkoandGnetophyta(Fig.1).Therehasbeen
steric restrictions of the side chains (Finkenstadt et al., a debate whether Gnetophyta are the gymnosperm
1995;Zhang etal.,2011). Howthen does the naturally lineagemostcloselyrelatedtotheangiosperms(Davis
occurring, highly substituted, xylan interact with cel- and Schaefer, 2011; Uddenberg et al., 2015). There are
lulose? Our recent findingsin the eudicot Arabidopsis few studies across gymnosperm lineages to determine
(Arabidopsisthaliana)revealedthatthemajorityofxylan anydivergenceinthestructureofxylans.
Figure 1. Schematic representation of the
phylogenetic relationship between gymno-
spermandangiospermspeciesstudiedinthis
work. The distances do not correspond to
phylogeneticdistances.
Plant Physiol. Vol. 171, 2016 2419
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Busse-Wicher et al.
Our work shows that some gymnosperm xylans
havedecorationsanddecorationpatternsthataredif-
ferent to those of eudicot xylans. Nevertheless, these
modifications largely reside on even xylosyl residues
on the backbone. Molecular dynamics simulations
support the hypothesis that this highly conserved or-
ganization of substitutions allows an unsubstituted
surface of xylan to bind stably to hydrophilic faces of
cellulosefibrils.
RESULTS
ConiferXylanIs DecoratedwithMeGlcA onEverySixth
XylosylResidue
Coniferxylanisdecoratedbya1,3-arabinofuranose
and a1,2-MeGlcA (Timell, 1967; Jacobs et al., 2001;
Willforetal.,2005;McKee,etal.,2016).Usingpartial
acid hydrolysis and MALDI-ToF mass spectrometry
(MS),Jacobsetal.(2001)analyzedthedistributionof
MeGlcA side chains on xylan from several conifers.
They suggested that the majority of substitutions
are regularly spaced, on every seventh or eighth
xylosyl residue along the backbone. A spacing of
seven residues would be incompatible with binding
to the hydrophilic surface of cellulose fibrils (Busse-
Wicheretal.,2016).Toinvestigatetheprecisepattern Figure2. MeGlcAisregularlydistributedonxylaninconifers.AIRof
of xylan MeGlcA substitution across plant lineages, Douglasfir,blackpine,commonjuniper,andtheeudicotArabidopsis
for comparison were hydrolyzed with glucuronoxylanase GH30
weusedaglucuronoxylanaseinCAZyfamilyGH30.
and analyzed by PACE. The major conifer products correspond to
Thisxylanasehydrolysesthexylanbackboneadjacent
substitutedxylohexosesUX andUAX.Undigestedmaterialwasused
to each MeGlcA, leaving MeGlcA at the O-2 linked asacontrol(2).Marker(M6),xylosylo6ligosaccharidesX-X;UX,X
position from the cleavage site (St John et al., 2011; 1 6 n n
substitutedbyMeGlcA.Bandsmarkedwithasterisksarenonspecific
Bromleyetal.,2013).Alcoholinsolubleresidue(AIR) labelingproducts.
fromdebarkedstemsofthreerepresentativeconifers
(Pinales: Douglas fir [Pseudotsuga menziesii], black
pine [Pinus nigra], and juniper [Juniperus communis])
ArabinoseandMeGlcAAreLocatedTwoXylosylResidues
were incubated with alkali to solubilize the hemicel-
ApartontheConiferXylanBackbone
luloses, and then digested with the GH30 xylanase.
The resulting oligosaccharides were analyzed by To determine any regularity in the position of the
PACE (polysaccharide analysis by carbohydrate gel arabinosyl side chains on the backbone, we analyzed
electrophoresis). In contrast to the range of oligosac- GH30 xylanase digestion products of alkali extracted
charides released from Arabidopsis xylan, just two (thus, all possible acetyl groups would have been re-
main oligosaccharides running between the stan- moved)xylanfromDouglasfir(conifer)byMS.MALDI-
dards Xyl and Xyl were produced from all three ToFMSrevealedtwomainionsofmasscorresponding
5 6
conifer xylans (Fig. 2). One of these oligosaccharides to MeGlcAPent (m/z 1023.3) and MeGlcAPent (m/z
6 7
comigrated with MeGlcAXyl (UX ), a product from 1155.3; Fig. 4), which is consistent with the PACE as-
6 6
digestion of Arabidopsis xylan. The other oligosac- signments of UX and UAX . No substitutions with
6 6
charide migrated slightly more slowly, suggesting unmethylated GlcA were detected. MALDI-TOF CID
an additional substitution. To investigate the na- MS/MS of 2-AB-labeled MeGlcAPent confirmed the
6
ture of the additional substitution, we digested oligosaccharide was UX , a linear pentose backbone
6
AIRfromDouglasfirwithGH30followedbyGH62 with 2-linked MeGlcA on the second xylosyl residue
a-arabinofuranosidase.Theoligosaccharidewassen- from the reducing end (Fig. 5). MS/MS of the MeGl-
sitive to the arabinosidase, indicating arabinose dec- cAPent peak showed similar fragmentation to UX
7 6
oration,andmigratedwithUX afterdigestion,sowe but indicated that an additional pentosyl side chain
6
nameditUAX (Fig.3).Takentogether,thedatashow substitution was located on the O-3 of the fourth Xyl
6
that the majority of MeGlcA substitutions are found from the reducing end (Fig. 5). Taken together with
oneverysixthxylosylresidueofconiferxylanandthe the a-arabinofuranosidase sensitivity (Fig. 3), we
xylan is additionally modified by a-arabinosyl side conclude that a-L-arabinofuranose is located at 22
chains. position from MeGlcA on the conifer xylan backbone
2420 Plant Physiol. Vol. 171, 2016
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Interaction of Xylan with Cellulose in Gymnosperms
and two representatives of the Gnetophyta (Ephedra
major and Gnetum edule). Digestion of AIR with GH30
xylanase and analysis by PACE showed that two oli-
gosaccharides were released from Cycad and Gingko
xylan,whichcomigratewiththetwooligosaccharides,
UX and UAX , released from the conifer Douglas fir
6 6
(Fig. 6). Further digestion with GH62 arabinofur-
anosidaseconfirmedtheseproductsareidenticaltothose
fromconiferxylan(Fig.7).Interestingly,UAX appearsto
6
be relatively more abundant than the UX , suggesting
6
greaterarabinosylationinGingkoandCycadsthaninthe
conifer xylan. In contrast to this, UAX was completely
6
absent in E. major (Gnetophyta) and UX was the pre-
6
dominant product (Fig. 6). The absence of arabinosyl
decorations was confirmed by insensitivity of the pro-
ducts to digestion with GH62 arabinofuranosidase
(Fig. 7). In addition to UX , we also detected some
6
degree of polymerization (DP) 8 and longer xylo-
oligosaccharides in E. major (Gnetophyta) (Figs. 6
and 7), indicating that there is a low proportion of
MeGlcA substitutions more widely spaced than
every sixth xylosyl residue. Nevertheless, six xylosyl
residues was the most frequent spacing of MeGlcA in
Gnetophyta.
Figure 3. a-Arabinofuranose decorations on conifer xylan. Glucur-
onoxylanaseGH30digestionproductsofDouglasfirweredigested
witha-arabinofuranosidaseGH62andanalyzedbyPACE.UAX was
6
sensitive to the arabinosidase, yielding UX . Marker (M), xylosyl
6
oligosaccharidesX -X .
1 6
(UAX ),maintaininganevenlyspacedpattern.PACE
6
andMSanalyseswererepeatedwithxylanfromblack
pine (conifer) and gave similar results (Supplemental
Figs.S1andS2).
XylanDecorationIsNotIdentical inDifferentLineages
ofGymnosperms
Xylan substitutions and substitution patterns are
conserved in different conifer species. However, the
Figure 4. Mass spectrometry of glucuronoxylanase GH30 digestion
xylan structure of the other gymnosperm lineages,
products of conifer xylan. A, MALDI-ToF-MS of glucuronoxylanase
Cycads, Gingko, and Gnetophyta, is unknown. To
GH30 digestion of NaOH-extracted, deacetylated Douglas fir xylan
investigate their xylan structures, we produced AIR
showstwospeciesUX andUAX.B,MALDI-ToF-MSofglucuronox-
6 6
from mature woody petioles of a Cycad (Encephalartos ylanase GH30 digestion of DMSO-extracted conifer xylan shows no
gratus)anddebarkedstemsfromGinkgo(Ginkgobiloba) acetylationofUX orUAX.
6 6
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Busse-Wicher et al.
Figure5. MALDI-CIDofglucuronoxylanaseGH30digestionproductsofconiferDouglasfirxylanshowsarabinoseandMeGlcA
substitutionsarespacedtworesiduesapart.A,High-energyMALDI-CIDoftheconiferUAX oligosaccharidelabeledwith2-AA
6
andseparatedbyHILIC.1,3-LinkedarabinoseispresenttwoxylosylresiduesfromtheMeGlcA.B,High-energyMALDI-CIDof
theconiferUX oligosaccharidelabeledwith2-AAandseparatedbyHILIC.
6
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Interaction of Xylan with Cellulose in Gymnosperms
suggesting possible acetylation (Fig. 8). MS of these
xylo-oligosaccharides confirmed the presence of acet-
ylation(Fig.8).Toinvestigatewhethertheacetylation
is patterned on alternate xylosyl residues, MS/MS
of the acetylated UX oligosaccharides was carried
6
out. Three acetylated UX products were detectable,
6
carrying the acetyl decorations precisely on alter-
nate residues (Fig. 9). Interestingly, acetylation was
not found on the xylosyl residues carrying MeGlcA,
unlikeeudicotxylanwherealmostallxylosylresidues
bearing MeGlcA are acetylated (Koutaniemi et al.,
2012).Moreover,analysisofxylanofG.eduleshowed
that this second Gnetophyta species has similar
MeGlcA and acetylation patterns (Supplemental Fig.
S4). Therefore, xylan in Gnetophyta is acetylated and
acetylationfollowsanevenlyspacedpattern.
XylanSubstitution PatterningintheMagnolialesIs
SimilartoThat ofEudicotGlucuronoxylan
The fact that in Gnetophyta we found eudicot-like
acetylation substitution patterns, but MeGlcA pat-
terns with characteristics of both conifers and eudi-
cots, prompted us to analyze xylan substitution in
Magnoliales, an early diverging angiosperm lineage.
The oligosaccharides produced by GH30 xylanase
digestion of magnolia (Magnolia x soulangeana; Fig. 6)
and bay laurel (Laurus nobilis; Supplemental Fig. S5)
xylan were highly similar to those from eudicots,
withaspacingof8mostfrequent,but6,10,12,and 14
spacing also substantial. The xylan was also acety-
latedandhasnoarabinosedecorations(Supplemental
Figure6. AnalysisoftheMeGlcAdistributiononxylaninthefour Fig. S6). Therefore, the Magnolialeshave a xylan deco-
gymnospermlineagesandMagnoliales.AIRpreparedfromrepre- rationpatternverysimilartoeudicots(Bromleyetal.,
sentativesofeachgymnospermlineage:Cycad(E.gratus),Ginkgo 2013;Derba-Maceluchetal.,2015).
(G. biloba), conifer (P. menziesii), and Gnetophyta (E. major)
and a representative of Magnoliales (Magnolia x soulangeana),
plustheeudicotArabidopsisforcomparison,werehydrolyzedby
MolecularDynamics Simulations ofXylanAdsorptionon
glucuronoxylanase GH30 and analyzed by PACE. The cycad, HexagonalCross-SectionCelluloseMicrofibrils
gingko, and conifer show UX and UAX , but the Gnetophyta
6 6
products are largely UX6. Marker (M), xylosyl oligosaccharides Insilicodockingofamodelofgymnospermxylan
X1-X6. onto a rectangular 24-chain cellulose fibril showed
that the even distribution of MeGlcA and arabinose
allows xylan adsorption on the hydrophilic (010)
InContrasttoOtherGymnosperms,theXylanof
and(020)surfaceswithoutsterichindrance(Fig.10).
GnetophytaIs Acetylated
This indicates gymnosperm xylan could bind to this
Acetylationofconiferxylanhasnotbeenreported. form of cellulose similarly to the glucuronoxylan
However, theGnetophyta xylan issimilar to second- from eudicots (Busse-Wicher et al., 2014). However,
arycellwallxylanineudicotsinthatitlacksarabinose the structure of the cellulose fibrils in eudicots and
substitution. This raised the question whether acety- gymnosperms is not resolved, and an alternative
lationmightbepresent,perhapsinordertomaintain hexagonal structure cellulose fibril is also consider-
solubilityofthexylanduringbiosynthesis.Toanalyze ed possible (Fernandes et al., 2011). The hydrophilic
theacetylationstatusofxylaninallfourgymnosperm facesofsuchcellulosefibrils(e.g.110)presentglucan
lineages,weextractedxylanusingDMSOtopreserve chainsavailableforhydrogenbondingtoxylan,but
acetylation and digested it with GH30 xylanase. the absence of surface grooves could lead to fewer
Douglas fir digestion products were not acetylated interplane glucan:xylan interactions and conse-
(Figs. 4 and 8) and neither were the products of quent instability in xylan chain binding. To inves-
ginkgo or cycads (Supplemental Fig. S3). In contrast tigate whether xylan could bind stably to cellulose
to this, the GH30 xylanase digestion products of fibrilswithahexagonalcrosssection,weperformed
E.major(Gnetophyta)weresensitivetoalkalitreatment, moleculardynamics(MD)simulationsofdecorated
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Busse-Wicher et al.
Figure7. Xylanisdecoratedwitha-arabinofuranose
in cycad and Ginkgo but not Gnetophyta.
GlucuronoxylanaseGH30digestionproducts
ofcycad(E.gratus;A),Ginkgo(G.biloba;B),
and Gnetophyta (E. major; C) xylan were
digested with a-arabinofuranosidaseGH62and
analyzed by PACE. UAX present in cycad and
6
GinkgoGH30digestionproductsissensitive
to the arabinosidase. Marker (M), xylosyl
oligosaccharidesX-X.
1 6
gymnosperm xylan chains adsorbed on the 110 hy- DP = 22 so that the (110) hydrophilic face was ex-
drophilic surface of an Ib cellulose microfibril. We posed to xylan adsorption. We considered the cases
builtamodelofconiferxylanwithbackboneDP14, of one and two xylan chains adsorbed onto the cel-
decorated with two a-1,2-linked glucuronic acids lulosesurface(Fig.11).Thesimulationsshowedthat
spaced by 6 xylosyl residues, and two a-1,3-linked the conifer xylan stably binds to the hydrophilic
arabinoses located two xylosyl apart from each GlcA cellulose surface (Supplemental Movies 1 and 2).
(Fig. 11). The cellulose microfibril was modeled Figure 12 show the two-dimensional distribution
as half of a hexagonal 36-chain elementary fibril of of xylan configurations defined by the distance of
Figure8. Analysisofxylanacetylationinco-
nifers and Gnetophyta. A, DMSO extracted
acetylxylans denoted as “Ac,” from Douglas
firandE.major(Gnetophyta)werehydrolyzed
withglucuronoxylanaseGH30.Afterenzyme
digestion and labeling with ANTS, some
samplesweresubjectedtoalkalitreatment
(NaOH) to remove any ester-linked acetyl
groups. A shift of the band mobility after
NaOHtreatmentindicatesthepresenceof
acetylation. Digestion products of AIR
deacetylated byalkali treatment, denoted as
“dAc,”areshownforcomparison.Undigested
material was used as a control. Marker (M),
xylosyl oligosaccharides X-X. B, MALDI-
1 6
ToF-MSofglucuronoxylanaseGH30digested
acetylatedxylanfromE.majorrevealsacety-
lation.Asteriskindicatesdoublysodiatedions
[M-H+2Na]+.
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Interaction of Xylan with Cellulose in Gymnosperms
Figure9. Gnetophytaxylanoligosaccharidesareacetylatedonalternatexylosylresidues.GlucuronoxylanaseGH30products
fromE.majorxylanwerelabeledwith2-AAandseparatedbyHILIC.High-energyMALDI-CIDofUXAc(A),UXAc (B),and
6 6 2
UXAc (C).ThepositionoftheacetateonthenonreducingXylisunknown.
6 3
xylosyl residues from the cellulose surface and by 1971). When two xylan chains are adsorbed in par-
the sum of the glycosidic torsion angles w+c, which allel with respect to one another and to the microfi-
characterizesthexylanhelicalconformation.Thefirst bril,thehelicalconformationalsofluctuatesaround
and last two residues were not considered in this w+c=120°, but with a more uniform distribution
analysis because they are considerably more flexible in the range w+c=90° to 150° (Fig. 12). In Figure
than the inner residues. Two-fold (2 ), right-handed 12C, we show the average values of w+cseparately
1
3-fold (3 ), and left-handed 3-fold screw conforma- for each glycosidic bond of the xylan backbone.
1
tions are characterized, respectively, by w+c=120°, Except for the residues belonging to the extremi-
w+c=50°, and w+c=190°. Figure 12 show that the ties of the xylan chain, the sum w+c alternates be-
distance between the xylosyl residues and the cellu- tween values somewhat above and below 120° for
lose surface remains mostly within 3 Å, indicating neighboringresidues,withthemeanrepeatdistance
stable binding of the xylan chains. Xylan-xylan in- of 10.4 Å (distance between bound xylobiose units),
teractionsstabilizethebindingfurther,asseenbythe indicating that the xylan chains do not exhibit
distribution of w+c, which are narrower for the two the idealized helical structure characterized by the
xylan chain case, indicating greater flexibility of the same values of w+c (120°) for all the residues of a
single chain. For the single conifer xylan, when the segment. Instead, the values of w+c from adjacent
xylosyl residues are close to the (110) cellulose sur- units average out to 120°, so that the adsorbed por-
face, the distribution of w+c is spread around 120° tions of the xylan chain adopts a 2 screw-like con-
1
with local maxima at 90° and 180° (Fig. 12). During formation. Thus, the alternating torsion angles
thefewtimeswhensomexylosylresiduesmoveaway provides a stretched conformation with all the sub-
from the cellulose surface, the helical conformation stitutions pointing away from the cellulose surface,
fluctuates strictly around the left-handed 3 screw allowing the xylan backbone to establish hydrogen
1
(w+c=190°),whichistheconformationobservedfor bonds with the cellulose fibril, as illustrated in
xylansinsolution(NieduszynskiandMarchessault, Figure 12.
Plant Physiol. Vol. 171, 2016 2425
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Busse-Wicher et al.
Figure 10. Model of conifer hemicellulose
interactions with hydrophilic surfaces
(010and020)ofa24-chaincellulosemicro-
fibril.Thereducingendviewandsidevieware
shown. DP 18 xylan chains, with evenly
spacedalternate(a-1-3)-arabinofuranosyland
(a-1-2)-MeGlcA decorations at xylosyl resi-
dues,weremodeledas2-foldhelicalscrews.
DISCUSSION MeGlcA pattern, acetylation and arabinosylation are
different across the gymnosperm lineages. Conifer,
Previously, little attention has been paid to the ar-
Cycad, and Ginkgo xylans are arabinosylated, but
rangement of substitutions along the xylan backbone
acetylation was not detected. In contrast, Gnetophyta
and the resulting implications for the interaction of
xylan is not arabinosylated but is acetylated, like the
the polysaccharide with cellulose. Here, we show that
xylansofeudicotsandtheearlydivergingangiosperms
precisepatterningofxylansubstitutionsiswidespread
suchasmagnolia.Therelationshipofthefourgymno-
in vascular plants. This xylan patterning is not re-
sperm and angiosperm lineages is to date still unre-
stricted to acetylation and MeGlcA substitutions, but
extendstoarabinosylresiduestoo.Weproposethatthe solved(DavisandSchaefer,2011).Thexylanstructural
patterning of xylan substitutions allows a compatible similarities place the Gnetophyta cell walls closer to
interactionbetweenxylanandthecellulosemicrofibril that of angiosperms than to the other gymnosperm
hydrophilic surfaces in vascular plants (Busse-Wicher lineages. Consistent with this, other cell wall compo-
etal.,2014;Busse-Wicheretal.,2016). nents in Gnetophyta are also more similar to angio-
ThepatternofMeGlcAsubstitutionofxylanissimi- sperm than gymnosperm cell walls. Conifers possess
larinthefourdifferentgymnospermlineages(Conifers, largelyGunitsinlignin,andthewallisdominatedby
Cycads,Ginkgo,andGnetophyta),withapredominant glucomannan.Incontrast,Gnetophytawallshaveboth
MeGlcAperiodicityofsix.Incontrasttotheconserved SandGunitsinlignin(Grabberetal.,2004),andxylan
Figure11. Cellulose-xylancomplexesused
for molecular dynamics simulations. A,
Coniferxylanmodel.B,Cellulosemicrofi-
brilwithoneandtwoconiferxylansdocked
ontothe(110)face.Onlythedark-graypart
ofthecellulosewasconsideredinthesim-
ulationstosavecomputationalcost.High-
lightedingreenaretheglucanchainsthat
werereplicatedandtranslatedtothecellu-
losesurfacetobuildthexylanchains.
2426 Plant Physiol. Vol. 171, 2016
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Interaction of Xylan with Cellulose in Gymnosperms
Figure12. Moleculardynamicssimulations
ofcellulose-xylancomplexes.Two-dimensional
distribution of the sum of dihedral angles
w+c of xylosyl residues against their dis-
tancesfromthecellulosesurfaceforone(A)
andtwo(B)coniferxylans.Thetwoxylosyl
residuesatbothendsofthexylanchainwere
not considered in the analysis. C, Average
valuesofw+cforeachglycosidicbondofthe
xylanbackbone.Theerrorbarscorrespond
to the standard deviations of the averages
taken from the three independent simula-
tions.D,Snapshottakenfromthesimulation
oftwoxylansadsorbedonthe(110)faceof
the cellulose fibril, showing the extended
configurationofthechain.
hashigherabundancethanglucomannan (Melvin and been fully assessed. In the commelinoid monocots, in-
Stewart,1969).Theseresultswouldtendtosuggestthat cluding grasses, xylans have acquired additional fea-
thecellwallsofancestorsofangiospermsandgymno- turessuchasferuloylation(CarpitaandGibeaut,1993;
sperms had these characteristics. Xylan acetylation Scheller and Ulvskov, 2010). In order to understand
wouldbelostandarabinosylationgainedintheconifer, xylan interaction with other cell wall components, it
ginkgo,andcycadgymnospermlineages. will be important to determine if cellulose-compatible
Patterning of arabinosyl substitution of cell wallxy- xylanstructuresexistintheseplantlineagestoo.
lanhasnotbeenpreviouslyreported.Weshowedthat There are still not enough data to support any sec-
the conifer xylan arabinosyl substitution is precisely ondary plant cell wall model, and there are multiple
twoxylosylbackboneresiduesfrom theMeGlcA.Fur- possibilitieshowandtowhatextentthecellulosefibers
thermore, acetylation resides only on every second may be covered and how cell wall components may
xylosylresidueinGnetophyta.Takentogether,wenow interact(HarrisandStone,2008).Havingacompatible
know that the majority of substitutions are evenly decorationpatternofMeGlcAandAra,cantheconifer
spaced in the xylan of gymnosperms as well as in xylanadsorbontocellulose?Insilicoexperiments(Fig.
Magnoliales and eudicots even though the modifica- 10)showedthatxylanfitstothe(020)/(010)hydrophilic
tions vary between lineages. Therefore, the even spac- faces of the proposed rectangular 24-chain fibril struc-
ing appears to be a highly conserved feature, strongly ture,similarlytotheundecoratedandacetylatedxylan
implyingabiologicalrole.Itisunknownifacompatible tested previously (Busse-Wicher et al., 2014). This cel-
pattern is present on the xylan of lower plant lineages lulose model, proposed by Fernandes et al. (2011) and
such as mosses, Selaginella, and ferns. Moreover, the Thomasetal.(2013),issupportedbyexperimentaldata.
decorationpatterninmonocotangiospermshasnotyet There are also alternative models proposed, including
Plant Physiol. Vol. 171, 2016 2427
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.