RESEARCH IN CONTEXT Evolution of mixed-linkage (1 3, 1 4)-b-D-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes Xinxin Xue † and Stephen C. Fry* The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JH, UK † Present address: The Biodiversity Research Centre, Department of Botany, Botanical Garden and Centre for Plant Research, The University of British Columbia, 2212 Main Mall, Vancouver V6T 1Z4, Canada. * For correspondence. E-mail [email protected]Received: 24 November 2011 Returned for revision: 20 December 2011 Accepted: 13 January 2012 Published electronically: 28 February 2012 † Background and Aims Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage (1 3, 1 4)-b-D-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicellu- loses occurring during Equisetum evolution. † Methods Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endogluca- nase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer chromatography. † Key Results Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide :disaccharide ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xylo- glucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosy- lated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides. † Conclusions G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the struc- ture and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit sizes. Key words: Equisetum, Hippochaete, Equisetum bogotense, ferns, eusporangiate monilophytes, leptosporangiate monilophytes, evolution, cell wall (primary), hemicellulose, mixed-linkage beta-glucan, xyloglucan. INTRODUCTION Major changes in primary cell wall composition often accom- panied landmark steps of plant evolution, especially during colonization of the land and vascularization (Stace, 1981; Kenrick and Crane, 1997a; Popper and Fry, 2003). Accordingly, a potential use of primary cell-wall composition in understanding phylogenetic relationships has been consid- ered, e.g. in algae (Popper and Fry, 2003), bryophytes (Ligrone et al., 2002; Popper and Fry, 2003, 2004), lycophytes and monilophytes (Buckeridge et al., 1999; Matsunaga et al., 2004; Popper and Fry, 2004) and angiosperms (Nothnagel and Nothnagel, 2007). Cell-wall components differ between plant taxa in parallel with their evolution and diversification (Popper, 2008) at both the monosaccharide and polysaccharide levels (Fig. 1). For instance, the earliest diverging extant vas- cular plants, lycopodiophytes, are unique among land plants in containing high levels of 3-O-methyl-D-galactose (Popper et al., 2001). To date, differences in cell-wall composition are commonly used as taxonomic markers in algal classifica- tion but have not been applied to land-plant classification (Stebbins, 1992; Buckeridge et al., 1999; Graham and Wilcox, 1999). The plant primary cell wall is a strong and cohesive network of cellulose microfibrils, probably tethered by hemicelluloses. Usually the principal tethers are xyloglucan (in dicots and non-commelinid monocots) or glucuronoarabinoxylan [in commelinid monocots, e.g. the Poaceae (grasses and cereals); Smith and Harris (1999)]. Structures and proportions of the hemicelluloses are variable between species and organs, and even within tissues (Harris, 2005; Fry, 2011). A third major hemicellulose, mixed-linkage (1 3, 1 4)-b-D-glucan (MLG), occurs in certain algae (Ford and Percival, 1965; Nevo and Sharon, 1969) including at least one charophytic species (Micrasterias denticulata; Eder et al., 2008), and in a narrow range of land plants (Stone # The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected]Annals of Botany 109: 873–886, 2012 doi:10.1093/aob/mcs018, available online at www.aob.oxfordjournals.org Downloaded from https://academic.oup.com/aob/article/109/5/873/141003 by guest on 28 December 2021
Transcript
RESEARCH IN CONTEXT
Evolution of mixed-linkage (1 � 3, 1 � 4)-b-D-glucan (MLG) and xyloglucanin Equisetum (horsetails) and other monilophytes
Xinxin Xue† and Stephen C. Fry*
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University ofEdinburgh, Daniel Rutherford Building, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JH, UK
†Present address: The Biodiversity Research Centre, Department of Botany, Botanical Garden and Centre for Plant Research,The University of British Columbia, 2212 Main Mall, Vancouver V6T 1Z4, Canada.
Received: 24 November 2011 Returned for revision: 20 December 2011 Accepted: 13 January 2012 Published electronically: 28 February 2012
† Background and Aims Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes inthe Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage(1 � 3, 1 � 4)-b-D-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported thatchanges in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicellu-loses occurring during Equisetum evolution.† Methods Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endogluca-nase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layerchromatography.† Key Results Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy ofextant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquelythe sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other speciesalso gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharideratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce inPsilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate fernsMarattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xylo-glucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosy-lated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides.† Conclusions G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied byquantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the struc-ture and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundanceof other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unitsizes.
Major changes in primary cell wall composition often accom-panied landmark steps of plant evolution, especially duringcolonization of the land and vascularization (Stace, 1981;Kenrick and Crane, 1997a; Popper and Fry, 2003).Accordingly, a potential use of primary cell-wall compositionin understanding phylogenetic relationships has been consid-ered, e.g. in algae (Popper and Fry, 2003), bryophytes(Ligrone et al., 2002; Popper and Fry, 2003, 2004), lycophytesand monilophytes (Buckeridge et al., 1999; Matsunaga et al.,2004; Popper and Fry, 2004) and angiosperms (Nothnagel andNothnagel, 2007). Cell-wall components differ between planttaxa in parallel with their evolution and diversification(Popper, 2008) at both the monosaccharide and polysaccharidelevels (Fig. 1). For instance, the earliest diverging extant vas-cular plants, lycopodiophytes, are unique among land plants incontaining high levels of 3-O-methyl-D-galactose (Popper
et al., 2001). To date, differences in cell-wall compositionare commonly used as taxonomic markers in algal classifica-tion but have not been applied to land-plant classification(Stebbins, 1992; Buckeridge et al., 1999; Graham andWilcox, 1999).
The plant primary cell wall is a strong and cohesive networkof cellulose microfibrils, probably tethered by hemicelluloses.Usually the principal tethers are xyloglucan (in dicots andnon-commelinid monocots) or glucuronoarabinoxylan [incommelinid monocots, e.g. the Poaceae (grasses andcereals); Smith and Harris (1999)]. Structures and proportionsof the hemicelluloses are variable between species and organs,and even within tissues (Harris, 2005; Fry, 2011). A thirdmajor hemicellulose, mixed-linkage (1 � 3, 1 �4)-b-D-glucan (MLG), occurs in certain algae (Ford andPercival, 1965; Nevo and Sharon, 1969) including at leastone charophytic species (Micrasterias denticulata; Ederet al., 2008), and in a narrow range of land plants (Stone
# The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
and Clarke, 1992; Buckeridge et al., 2004; Trethewey et al.,2005; Fry et al., 2008a; Sørensen et al., 2008). Within landplants, MLG has been found in only two, distantly related,groups: the angiosperm order Poales (Smith and Harris,1999; Buckeridge et al., 2004; Trethewey et al., 2005) andthe ‘fern ally’ genus Equisetum (Fry et al., 2008a; Sørensenet al., 2008). Since MLG occurs in two evolutionarily verydistant land-plant taxa, it appears that its biosynthesis iseither deeply conserved or has arisen twice through convergentevolution (Sørensen et al., 2010).
Although the polysaccharide MLG is found in both thePoales and the Equisetales, only the latter possess MLG : xylo-glucan endotransglucosylase (MXE), an enzyme capable ofgrafting MLG to xyloglucan chains by transglycosylation(Fry et al., 2008b). This observation may indicate that MLGserves different biological roles in the cell walls of these twoplant orders. A difference in role is also suggested by thefact that poalean MLG is principally a feature of young,rapidly expanding tissues, whereas the MLG of Equisetum per-sists and may even increase during ageing (Fry et al., 2008a).MXE activity reaches its peak in old, tough Equisetum stems,suggesting a role in tissue strengthening (Fry et al., 2008a).
MLG is a long, unbranched, coiling b-D-glucan chain with(1 � 4) linkages plus a minority of (1 � 3) linkages. In
cereals, MLG is synthesized in the Golgi apparatus (Carpitaand McCann, 2010) and typically has a structure of the type. . . G4G4G3G4G4G3G4G4G4G3G4G4G3G4G4G3 . . .where G represents a b-D-glucose residue, and 3 and 4 repre-sent (1 � 3) and (1 � 4) bonds, respectively. The underlinedsegments are effectively cello-oligosaccharides interconnectedby ‘hinges’. The (1 � 4) linkages give rigidity, whereas (1 �3) linkages confer flexibility and water solubility (Woodwardet al., 1983). Luttenegger and Nevins (1985) found a markedvariation in the MLG content (1–14 %) of Zea mays coleoptileprimary cell walls during changes in the rate of cell expansion,suggesting a role of MLG in rapid growth in the Poales.
MLG can be structurally characterized by analysis ofthe oligosaccharides produced on digestion with Bacillussubtilis lichenase. This commercial endo-glucanase cleavesthe (1 � 4) bond following a (1 � 3) bond (Meikle et al.,1994). From poalean MLG, lichenase thus generates the tri-saccharide G4G3G plus a minority of the tetrasaccharideG4G4G3G (Stone and Clarke, 1992) (these sequences arealways quoted from non-reducing to reducing end).
While poalean MLG is composed of G4G3G . G4G4G3G,Equisetum MLG when digested with lichenase releasesG4G4G3G and the disaccharide G3G (laminaribiose) as thepredominant products (Fry et al., 2008a; Sørensen et al.,
Streptophytes
Embryophytes
Tracheophytes
Euphyllophytes
Seed plants
Angiosperms
Monosaccharide residues
MeRha
MeGal
Polysaccharides
Xyloglucan
Mannans
Xylan
RG-II
MLG
Homo-GalA
Monilophytes
Cha
roph
ytes
Incr
easi
ngab
unda
nce
Bry
ophy
tes
Lyco
phyt
es
Eus
pora
ngia
tem
onilo
phyt
es
Lept
ospo
rang
iate
fern
s
Gym
nosp
erm
s
Com
mel
inid
mon
ocot
s
Non
-com
mel
inid
mon
ocot
s
Dic
ots
FI G. 1. Scheme of streptophyte phylogeny, shown together with the relative abundance of selected primary cell wall components across major plantgroups. Wall composition differs at both the monosaccharide and the polysaccharide levels. MeGal, 3-O-methyl-D-galactose; MeRha, 3-O-methylrhamnose;MLG, (1 � 3, 1 � 4)-b-D-glucan; RG-II, rhamnogalacturonan-II; Homo-GalA, homogalacturonan. Intensity of the shades indicates relative abundance.
MLG (dotted shading) is present in only one genus (Equisetum) of eusporangiate ferns and one order (Poales) of the commelinid monocots.
Xue & Fry — Monilophyte hemicelluloses874
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
2008). The trisaccharide : tetrasaccharide ratio in poaleanMLG ranges between 1.5 in Zea mays and 4.5 in wheat(Triticum aestivum) flour (Buckeridge et al., 1999; Li et al.,2006). In addition to G4G3G and G4G4G3G, oligosaccharideswith degree of polymerization (DP) up to 14 have beenreported in barley MLG (Lazaridou and Biliaderis, 2007);and Equisetum MLG yielded a lichenase product tentativelyidentified as DP9 (Fry et al., 2008a).
Functionally, the trisaccharide : tetrasaccharide ratio affectsthe strength of an MLG gel, as demonstrated by studiesusing the large-deformation mechanical test (Lazaridouet al., 2004), suggesting a significance of this ratio ingoverning the strength of the primary cell wall.
In the primary cell walls of many land plants, except thecommelinid monocots, xyloglucan is the major hemicellulose.Composed of long and slightly flexible chains, xyloglucanscan hydrogen-bond strongly to cellulose, and probably tetheradjacent microfibrils, contributing to wall architecture.Xyloglucans have a backbone of (1 � 4)- b-D-glucan (qualita-tively identical to cellulose), many of the glucose residues(typically approx. 75 %) bearing an a-D-xylopyranose (Xylp)residue at position 6. A (1 � 4)-linked glucose is abbreviatedG; one with xylose attached (forming a disaccharide calledisoprimeverose) is abbreviated X (Fry et al., 1993).Additional sugar residues are attached to some of theisoprimeverose groups, generating structures such as b-D-Galp-(1 � 2)-a-D-Xylp-(1 � 6)-b-D-Glc (abbreviated L)and a-L-Fucp-(1 � 2)-b-D-Galp-(1 � 2)-a-D-Xylp-(1 � 6)-b-D-Glc (abbreviated F).
Xyloglucan can be broken down for analysis by digestionwith xyloglucan endoglucanase (XEG; Pauly et al., 1999),which yields oligosaccharides such as the heptasaccharideXXXG, the two octasaccharides XXLG and XLXG, the twononasaccharides XXFG and XLLG, and the decasaccharideXLFG (Hoffman et al., 2005).
The monilophyte Equisetum hyemale and the lycophyteSelaginella kraussiana have been reported to possess addition-al xyloglucan repeat-units not yet detected in other plants.These are D and E, which are identical to L and F, respective-ly, except that the b-D-Galp residue is replaced by a-L-Arap(Pena et al., 2008). This is a highly conservative replacement:despite the a/b and D/L differences in the nomenclature,b-D-Galp and a-L-Arap differ from each other only in thefact that the former has a -CH2OH group in place of an -H.Equisetum hyemale xyloglucan includes the sequencesXLEG and XLDG, which very closely resemble XLFG andXLLG, respectively (Fig. 2).
Extant vascular plants are divided into two major clades:lycophytes and euphyllophytes, the latter (plants with ‘true’leaves) being divided into spermatophytes and monilophytes(Kenrick and Crane, 1997b; Pryer et al., 2001). The horsetails(genus Equisetum), the psilophytes, and the eusporangiate andleptosporangiate ferns, are all monilophytes (Pryer et al., 2001,2004). The class Equisetopsida (syn. Sphenopsida) emerged inthe Upper Devonian, and became diverse and abundant in thePalaeozoic swamp forest (Delevoryas, 1962). It includes boththe extant genus Equisetum and extinct herbaceous and arbor-escent horsetails such as the Calamitaceae (Bateman, 1991).The extant herbaceous horsetails are considered ‘livingfossils’ as Equisetum is the only surviving genus, forming a
crown group (i.e. a clade including all the extant membersfrom their most recent common ancestor) of theEquisetopsida total group (a clade containing all descendantsof a common ancestor, whether extant or extinct). Equisetummight even be the oldest surviving genus of all vascularplants owing to the ancient history of this lineage (Hauke,1978).
Both molecular- and fossil-based estimates of age suggestthat extant Equisetum had diverged from the fossil genusEquisetites in the Tertiary of the Cenozoic (approx. 49 Ma)(Des Marais et al., 2003; Pryer et al., 2004). However,recent comparisons of several anatomically preservedEquisetum fossils from the Jurassic and Cretaceous period in-dicate a much earlier Mesozoic origin of the crown group(approx. 100–200 Ma) (Stanich et al., 2009; Channing et al.,2011). This argument is based on the distinct morphologicalfeatures found in Mesozoic Equisetum (from sinter and othervolcanic deposits), which closely resemble but differ fromboth the Triassic Equisetites fossil compressions and theextant horsetails (Baron, 1889; Channing et al., 2011).
Leaves of Equisetum have the combined characteristics ofapparent microphylls (leaves with a single vascular bundle)plus the megaphyll feature of having a gap in the stem vascu-lature where the leaf bundle diverges. The fossil evidence isclear that the apparent microphylls of Equisetum actually rep-resent reduced megaphylls, based on observations from thegenus Sphenophyllum in the sister group Sphenophyllales,which have more complex megaphylls from which type theleaves of Equisetum are apparently reduced (Kenrick andCrane, 1997b).
Recent phylogenetic analysis of the 15 recognized speciesof the genus Equisetum shows a basal trichotomy: there aretwo monophyletic subgenera [Hippochaete (including the neo-tropical E. giganteum and E. myriochaetum) and Equisetum(all of which are of temperate distribution in the northernhemisphere)] plus the diminutive neotropical speciesE. bogotense as a sister group (Hauke, 1978; Des Maraiset al., 2003). [Note: ‘Equisetum’ in this manuscript impliesthe genus unless ‘subgenus’ is specified.] Species in the sub-genus Equisetum typically have superficial stomata withhighly branched vegetative shoots, whereas most species inHippochaete have sunken stomata with generally unbranchedvegetative shoots (Hauke, 1959).
Phylogenetic study of Equisetum is difficult owing to thelong history of isolation and frequent inter-specific hybridiza-tion within but not between each subgenus. In addition, horse-tails are capable of reproducing vegetatively, furthercomplicating phylogenies as sterile hybrids can persist.While the phylogenetic relationships among species ofHippochaete have shown a fair degree of consistency acrossstudies (Des Marais et al., 2003; Guillon, 2004, 2007), thephylogeny of subgenus Equisetum is still disputable, despitethere being more distinguishing morphological featureswithin the group than in Hippochaete. One suggested reasonfor the lack of reconciliation between molecular and morpho-logical classification is the homoplasic characters (such asstem dimorphism), arising through convergent evolution asanalogous features, not being differentiated in classical taxo-nomic treatments (Guillon, 2004, 2007). The systematic diffi-culty of Equisetum is not unique in the early-divergent
Xue & Fry — Monilophyte hemicelluloses 875
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
lineages of euphyllophytes: there is a significant morphologic-al gap between ferns and seed plants as a result of age, extinc-tion and lack of fossil record (Pryer et al., 2001, 2004).
The objectives of the present work were to explore the oc-currence and abundance of MLG in diverse monilophytes,and to define MLG structures from both horsetail subgeneraand E. bogotense to test whether differences arose during thecourse of evolution in the genus Equisetum. We also aimedto develop a simple visual method for surveying the quantita-tive and qualitative variation occurring in xyloglucan through-out the monilophytes.
MATERIALS AND METHODS
Plant materials
Plant sources are listed in Table 1. Most fresh plants wereobtained from the Royal Botanic Garden Edinburgh (RBGE)living collection with accession numbers indicated, with theexception of E. arvense and E. fluviatile which were collectedfrom the wild in Edinburgh. Vouchers of E. bogotense werecourtesy of New York Botanic Garden; four independent herb-arium specimens were combined to make an adequate sample.In the genus Equisetum (except for the herbarium specimen),
DP10
DP8
Glc
Glc
GlcGlc
Glc
GlcXyl
Glc
Glc
GlcXyl
Glc Xyl
XylAraFucG
al
GlcRT GlcRT
GlcRTGlcRT
Gal
XylX
yl
Xyl
Gal
Gal
Fuc
Xyl
Ara
Glc
Glc
Xyl
Xyl Xyl
Xyl
XLFG XLEG
XXLG XXDG
CH3
CH2
CH2
CH2
CH2OH
CH2OH CH2OH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2OH
CH2
CH2
CH2
CH2
CH2
CH3CH2
HO
HO HOOH
HOHO
O O
O OO
O
OO
O O
O
O
O
OOH
OH OH
OH
OH
OH OHOH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OHHO
HO
HO
HO
HO
HO
HO HO
HO
HO
HO
HO
HO
HO
HO
O OO
O
OO
O
O
O
O
O
O
O O
O
OO
O
O
O
OO
O
O
OO
O
O O
O
OH
OHHO
HO
HO
HO
O
OH
OH
OHOH
OH
HO
HO
HO
HO
HO
OH OH
OH
OH
OH
OH
OH
OH
OH
HO
HOOH
OHOH
OH
OH
OH
OH
OH
HO
HO
OHOH
OH
OH
OH
HO O O
O
O
O O
OO
O
O
O O
O
O
OOO
O
O
OO
O
O
FI G. 2. Representative xyloglucan oligosaccharides. Top, decasaccharides; bottom, octasaccharides; left, structures found in most vascular plants; right, struc-tures detected by Pena et al. (2008) in Equisetum hyemale and Selaginella kraussiana. Note the minor chemical difference (grey ovals) between the left-hand andright-hand structures. The TLC system shown in Fig. 7 would be expected to group the two similar decasaccharides together and the two similar octasaccharidestogether. Sugar residues are labelled in grey: Ara, a-L-arabinopyranose; Fuc, a-L-fucopyranose; Gal, b-D-galactopyranose; Glc, b-D-glucopyranose; Xyl,
a-D-xylopyranose. GlcRT indicates the reducing terminal glucose group.
Xue & Fry — Monilophyte hemicelluloses876
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
TABLE 1. Classification, sources, AIR yields, and relative abundance of MLG subunits and xyloglucan of all species analysed
Classification SpeciesSource/accession
number* Age†Polymer content
(mg g21)‡
Relative oligosaccharide yields§
MLG4 MLG3 MLG2 XGOs
HETEROSPOROUS LYCOPHYTESelaginellaceae Selaginella willdenowii (Desv. ex Poir.)
Baker19762969(E) Y 117 – – – +
EUSPORANGIATE MONILOPHYTESOphioglossaceae Ophioglossum vulgatum L. 19695522(E) Y 67 – – – +Psilotaceae Psilotum nudum (L.) P. Beauv. 20040835(E) Y 45 – – – +
Equisetum ramosissimum ssp. debile Hauke 19731694(E) M nd + – ++ + [ ]Marattiaceae Marattia fraxinea Sm. 19697184(E) Y 68 – – – ++ +
M 65 – – – ++ +Angiopteris evecta Hoffm. 20060955(E) Y 82 – – – ++ +
M 58 – – – ++Angiopteris lygodiifolia Rosenstock 19763705A(E) Y 29 – – – ++ +
M 57 – – – ++ +LEPTOSPORANGIATE MONILOPHYTESOsmundaceae Osmunda regalis L. 19913878A(E) Y 108 – – – ++ +
M 203 – – – +Todea barbara (L.) T. Moore 19652792(E) Y 98 – – – ++ +
M 134 – – – +Hymenophyllaceae Trichomanes speciosum Willd. 19992144(E) Y 191 – – – ++ +
M 224 – – – ++Dipteridaceae Dipteris conjugata Reinw. 20021886A(E) Y 145 – – – +
M 249 – – – +Lygodiaceae Lygodium japonicum (Thunb.) Sw. 19734355(E) Y nd – – – ++
M nd – – – +Anemiaceae Anemia sp. 19933657(E) Y 116 – – – ++ +
M 154 – – – ++Marsileaceae Marsilea drummondii A. Braun 19933710(E) Y 100 – – – +
M 111 – – – +Thyrsopteridaceae Thyrsopteris elegans Kunze 20031267A(E) Y 108 – – – ++ +
M 70 – – – +Cyatheaceae Cyathea spinulosa Wall. 19941397A(E) Y 87 – – – ++ +
M 54 – – – ++Woodsiaceae Woodsia obtusa (Spr.) Torrey 20061094A(E) Y 99 – – – ++ +
M 73 – – – ++SPERMATOPHYTESGymnosperm Chamaecyparis lawsoniana Parl. KB Y 216 – – – ++
M 255 – – – ++Angiosperm Nymphaea lotus L. 20040381(E) Y 26 – – – ++ +
M 278 – – – ++ +
* Sources/accession numbers: (E), Royal Botanic Garden, Edinburgh (RBGE) accession no.; KB, King’s Buildings campus, Edinburgh; NYBG, New YorkBotanic Garden.
† Age of tissues tested: Y, young tender tissues; M, mature tough tissues; H, herbarium tissue.‡ Polymer contents: yield of alcohol-insoluble residue (mg AIR g21 fresh weight); nd, not determined.§ Oligosaccharides indicative of MLG and xyloglucan: MLG4, MLG3, MLG2, tetra-, tri- and disaccharide of MLG released by lichenase; XGOs,
xyloglucan oligosaccharides released by XEG. Yields of the oligosaccharides are scored as: –, undetectable; +, inconsistently or barely detectable; +, ++ ,+ ++ , low, moderate and high abundance; [ ], not determined.
} E. bogotense cannot be placed clearly in either subgenus.
Xue & Fry — Monilophyte hemicelluloses 877
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
young tender tissues were taken from the base of each inter-node wrapped inside the leaf sheath; mature tissues were thetougher parts of internodes. In other genera, young (tender)and mature (tough) tissues were selected empirically.
Preparation of alcohol-insoluble residue (AIR) andhemicellulose B
Methods followed previous protocol (Fry, 2000). AIR wasobtained by homogenizing plant materials in 96 % ethanol, fil-tering on Miracloth (Calbiochem, EMD, USA), then repeated-ly rinsing with 96 % ethanol until all the chlorophyll had beenremoved and the filtrate was colourless. After drying at roomtemperature, AIR (100 mg) was shaken at 37 8C for 17 hwith 5 mL 6 M NaOH containing 0.1 % NaBH4. The super-natant was neutralized with acetic acid and the suspensionwas dialysed in 12–14-kDa cut-off tubing (Medicell Int.Ltd, London, UK) against running tap-water for 3 d. The sus-pension from inside the sac was centrifuged at 6000 g for20 min. Both the pellet (hemicellulose A) and the supernatant(hemicellulose B) were retained for analysis.
Lichenase digestion
Hemicellulose was digested with lichenase (from Bacillussubtilis; Megazyme), yielding MLG repeat-units, as describedby Popper and Fry (2003). Hemicellulose solution [200 mL,0.5 % w/v in pyridine/acetic acid/chlorobutanol/water (1 : 1 :0.5 : 98, v/v/w/v; PyAW)] was mixed with 200 mL lichenasesolution (1.43 unit mL21) and incubated at 20 8C with gentleshaking for 16 h. Digestion was then stopped with 200 mLformic acid. The products were dried, re-dissolved in 150 mLPyAW, and analysed by thin-layer chromatography (TLC).
Xyloglucan endoglucanase (XEG) digestion
XEG (a gift from Novo Nordisk, Bagsværd) fromAspergillus aculeatus contained traces of pectinase andb-galactosidase. Conditions for XEG digestion, with minimalside reactions, were devised in preliminary studies, withcitrus pectin and tamarind seed xyloglucan as substrate,monitoring galacturonic acid and galactose production,respectively.
For the XEG digestion, 200 mL of sample (0.5 % w/v; sus-pension of hemicellulose A or solution of hemicellulose B) inPyAW was mixed with 200 mL of 0.05 % (w/v) XEG in PyAWand incubated at 25 8C for 64 min. The reaction was stoppedwith 200 mL formic acid. Products were dried and re-dissolvedin 150 mL water; 3 mL was analysed by TLC.
Thin-layer chromatography
Samples were loaded on to a TLC plate (Merck, silicagel 60, 20 × 20 cm; Sigma-Aldrich, UK) and developed ineither butanol/acetic acid/water (2 : 1 : 1, v/v/v) or butanol/ethanol/water (2 : 1 : 1, v/v/v), as specified in the figurelegends. Sugars were stained with thymol (Frankova and Fry,2011).
RESULTS
Preparation of alcohol-insoluble residue
Samples of young and mature tissues from 27 species, mainlymonilophytes, were converted to alcohol-insoluble residue(AIR; i.e. total polymers, a high proportion of which will becell wall material). The species investigated are listed inTable 1, and most are illustrated in Fig. 3. There was consid-erable variability between species in total polymer contentper gram fresh weight (Table 1), reflecting wide variation inthe species’ adaptations to diverse environments (wet anddry, exposed and sheltered, insolated and shaded, etc.).However, data for all 21 monilophyte species in which bothmature and young tissues were quantified show a mature :young ratio (of mg AIR obtained per g fresh weight) of1.42+ 0.15 (mean+ s.e., N ¼ 21), supporting our subjectivedescription of these samples as ‘tough’ and ‘tender’, respect-ively. The mature tissues are likely to be richer in secondarycell wall material, deposited after the cells had lost theability to expand.
Mixed-linkage (1 � 3, 1 � 4)-b-D-glucan (MLG)
Hemicelluloses from AIR of numerous species of monilo-phyte plus a representative lycophyte and two spermatophyteswere tested for MLG by TLC analysis of lichenase digests.Hemicellulose A (i.e. the alkali-extracted material that precipi-tates when neutralized) gave almost no MLG oligosaccharides(chromatograms not shown). On the other hand, the hemicellu-lose B (i.e. that which remains in solution after neutralization)from certain species gave a series of oligosaccharides diagnos-tic of MLG. Production of these oligosaccharides was depend-ent on the presence of lichenase (Fig. 4), confirming that theywere not due to artifactual degradation of polysaccharides. Asummary of all the following MLG oligosaccharide data isgiven in Table 1.
All species of the genus Equisetum gave MLG oligosacchar-ides, but in varying yields (Fig. 4). The major product in mostEquisetum species was the tetrasaccharide G4G4G3G, in con-trast to the situation with poalean MLG (e.g. fully lichenase-digested commercial barley MLG; Fig. 4), which gavemainly the trisaccharide G4G3G. There was little consistentdifference, either quantitative or qualitative, between youngand mature tissues. No non-Equisetum monilophytes, eithereusporangiate or leptosporangiate, gave detectable MLG oligo-saccharides (Fig. 4 and all other species listed in Table 1; chro-matograms not shown). Likewise, no MLG was detected in thelycophyte Selaginella willdenowii or either of the non-commelinid spermatophytes examined (results not shown).
Samples rich in starch may give traces of malto-oligosacchar-ides on lichenase digestion owing to slight contamination of theenzyme with amylases. Therefore, digests were analysed byTLC in two different solvent systems, with amalto-oligosaccharide marker ladder. MLG oligosaccharideswere confirmed to be present in all Equisetum samples(Fig. 5). Somewhat different patterns were noted among the pro-ducts generated from the nine surveyed species of horsetail. Ingeneral, there was a higher concentration of MLG in the hemi-cellulose of subgenus Equisetum and E. bogotense than in
Xue & Fry — Monilophyte hemicelluloses878
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
A B C D
E E¢ F G H I
J K L M M¢
M¢¢ N O P Q
R S S¢ T U V
V¢ W X Y Z
FI G. 3. Plant specimens analysed in the present work. (A) Selaginella willdenowii; (B) Equisetum × bowmanii; (C) Equisetum × robertsii; (D) Equisetumarvense; (E, E′) Equisetum fluviatile; (F) Equisetum sylvaticum; (G) Equisetum pratense; (H) Equisetum hyemale var. affine; (I) Equisetum myriochaetum;(J) Ophioglossum vulgatum; (K) Psilotum nudum; (L) Marattia fraxinea; (M, M′, M′′) Angiopteris evecta; (N) Angiopteris lygodiifolia; (O) Osmundaregalis; (P) Todea barbara; (Q) Dipteris conjugata; (R) Trichomanes speciosum; (S, S′) Lygodium japonicum; (T) Anemia sp.; (U) Marsilea drummondii;(V, V′) Thyrsopteris elegans; (W) Cyathea spinulosa; (X) Woodsia obtusa; (Y) Chamaecyparis lawsoniana; (Z) Nymphaea lotus. Extraneous species, e.g.the pink-flowering Geranium seen in (D), were removed prior to analysis of the plants of interest. Letters A–Z are colour-coded to agree taxonomically with
the labelling on Figs 4–7.
Xue & Fry — Monilophyte hemicelluloses 879
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
subgenus Hippochaete. In some samples of E. hyemale MLGwas almost undetectable.
The trisaccharide G4G3G (the major repeat-unit in poaleanMLG) was consistently a very minor component of EquisetumMLG. The tetrasaccharide G4G4G3G and disaccharide G3G(¼ laminaribiose) were the major repeat-units. There was noconsistent difference between the subgenera in tetrasacchar-ide : disaccharide ratio. In some samples from both subgenera(e.g. some samples of mature E. arvense and matureE. ramosissimum; Fig. 5), the disaccharide predominatedover the tetrasaccharide; but in other samples the tetrasacchar-ide was predominant (Fig. 6). The very isolated (probablyearliest-diverging) species E. bogotense uniquely had MLGcomposed almost solely of the tetrasaccharide.
Xyloglucan
Xyloglucan-derived oligosaccharides were almost undetect-able in digests of hemicellulose A (chromatograms not shown).Therefore, only hemicellulose B was analysed in detail. Asummary of the yields of hemicellulose B xyloglucan-derivedoligosaccharides is given in Table 1. As expected, xyloglucantended to be more abundant in the hemicellulose B of youngtissues than of mature (Fig. 7), the former being richer inprimary cell walls. Among the eusporangiate monilophytes,total xyloglucan was scarce in the horsetail subgenus Equisetumand in Psilotum nudum, but abundant in the horsetail subgenusHippochaete and in the ferns Marattia and Angiopteris. The lep-tosporangiate ferns varied widely in xyloglucan content: in some(Osmunda, Todea, Trichomanes, Anemia, Thyrsopteris, Cyatheaand Woodsia) it was abundant, whereas in others (Dipteris,Lygodium and Marsilea) it was scarce (Fig. 7).
In all cases where the yield of xyloglucan oligosaccharides wassufficient for a TLC profile to be clearly discerned, a ‘corepattern’ of five classes of oligosaccharide repeat-unit was
similar across the eusporangiate and leptosporangiate monilo-phytes, resembling that in the seed-plants Chamaecyparus andNymphaea. The major repeat-unit classes co-migrated withXLFG, XLLG, XXFG, XXLG (and/or XLXG), and XXXG(DP 10, 9NF, 9F, 8 and 7, respectively, where NF and F indicatenon-fucosylated and fucosylated), giving a characteristic ‘‖-‖--|’pattern, where ‘|’ represents a band.
There was taxonomic variation in the intensity of the fivebands in the core ‘‖-‖--|’ pattern of xyloglucan oligosacchar-ides. For example, Marattia, Angiopteris, E. hyemale andNymphaea exhibited relatively high intensities of the DP9NF
fragment. In addition, many species also exhibited a probableDP6 band running slightly faster than XXXG.
A discordant selection of species, upon XEG digestion, alsogave two major, unidentified oligosaccharides that appeared tobe a di- and trisaccharide of glucose. After purification by gel-permeation chromatography, they gave no isoprimeverose onDriselase digestion, and only glucose on acid hydrolysis. OnTLC, the disaccharide and trisaccharide migrated slightlyslower than cellobiose and cellotriose, respectively (markerchromatograms, not shown). The disaccharide was also distin-guished chromatographically from authentic maltose, isomal-tose and gentiobiose, and remains an unidentified product ofXEG action. The lack of xylose, and non-identity withcello-oligosaccharides, suggests that the two small oligosac-charides were not derived from any known xyloglucan struc-ture. They were particularly abundant in XEG digests of theeusporangiate fern Angiopteris, two leptosporangiate ferns(Osmunda and Lygodium), and the gymnospermChamaecyparis, but were not abundant in any Equisetumspecies nor in Nymphaea, an early-diverging dicot nested inthe basal ‘ANITA’ (Amborella, Nymphaea, Illicium,Trimenia and Austrobaileya) grade according to APGII (2003).
Hemicellulose B preparations from the tested species gavewidely varying yields of glucose on XEG digestion, and
Mar
kers
a
Mar
kers
b
E. a
rven
se Y
–
E. a
rven
se Y
+
E. a
rven
se M
–
E. a
rven
se M
+
E. f
luvi
atile
Y–
E. f
luvi
atile
Y +
subgenusEquisetum
subgenusHippochaete
subgenusEquisetum
Eusporangiatefern
Leptoporangiatefern
E. h
yem
ale
Y–
E. h
yem
ale
Y +
E. m
yrio
chae
tum
Y–
E. m
yrio
chae
tum
Y +
E. m
yrio
chae
tum
M–
E. m
yrio
chae
tum
M+
E. p
rate
nse
Y–
E. p
rate
nse
Y +
E. p
rate
nse
M–
E. p
rate
nse
M+
Mar
kers
c
Mar
kers
a
Mar
kers
b
E. l
itora
le Y
–
E. l
itora
le Y
+
E. r
ober
tsii
Y–
E. r
ober
tsii
Y+
E. r
ober
tsii
M–
E. r
ober
tsii
M+
Ang
iopt
eris
lygo
diifo
lia Y
–
Ang
iopt
eris
lygo
diifo
lia Y
+
Ang
iopt
eris
lygo
diifo
lia M
–
Ang
iopt
eris
lygo
diifo
lia M
+
Osm
unda
reg
alis
M
Osm
unda
reg
alis
M+
Tode
a ba
rbar
a Y
–
Tode
a ba
rbar
a Y
+
T o
dea
barb
ara
M–
Tode
a ba
rbar
a M
+M
arke
rs c
Glc
Lam2
MLG3
MLG4
MLG6MLG7
M2
M4
M7
Glc
M2
M4
M7
FI G. 4. Lichenase digestion products of hemicellulose B from several monilophytes, with enzyme-free controls. Digests of: Y, young tender tissues; M maturetough tissues; ‘ + ’ lichenase-treated; ‘–’, enzyme-free. The TLCs were developed in butanol/acetic acid/water, and stained with thymol. Markers a and b: oli-gosaccharides of DP 3–7 obtained by partial (a) and complete (b) lichenase digestion of commercial barley MLG (MLG3, G4G3G; MLG4, G4G4G3G; MLG6,G4G3G4G4G3G; MLG7, G4G4G3G4G4G3G and/or G4G3G4G4G4G3G). Markers c: M2, maltose; M4, maltotetraose; M7, maltoheptaose. Laminaribiose
(Lam2) was detectable in some digests. Spots marked ‘*’ are contamination, not seen on replicate plates.
Xue & Fry — Monilophyte hemicelluloses880
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
there was a tendency for glucose to correlate with the yield ofthe small oligosaccharides mentioned in the previous para-graph. Several authentic polysaccharides (including starch,
lichenan and yeast glucan) gave small amounts of glucoseon digestion with XEG under the conditions used. Therefore,the glucose detected was not necessarily of xyloglucan origin.
1 G
luco
se
2 M
alto
-ladd
er
3 F
estu
ca a
rund
inac
ea
4 E
. arv
ense
Y
5 E
. arv
ense
M
6 E
. flu
viat
ile M
7 E
. syl
vatic
um Y
8 E
. x b
owm
anii
Y
9 E
. rob
erts
ii Y
10 E
. hye
mal
e Y
11 E
. hye
mal
e M
12 E
. myr
ioch
aetu
m Y
13 E
. myr
ioch
aetu
m M
14 E
. ram
osis
sim
um M
15 E
. bog
oten
se H
16 L
amin
arib
iose
17 C
ello
bios
e
18 M
alto
se
19 G
entio
bios
e
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Glc
BEW
BAW
Lam2
Mlt2
MLG3
A
B
MLG4
Mlt3
Mlt4
Mlt5
Mlt6
Mlt7Mlt8Mlt9
Mlt10
Glc
Lam2Mlt2
MLG3
MLG4
Mlt3
Mlt4
Mlt5
Mlt6
Mlt7
Mlt8
Mlt9Mlt10
Markers
Poales
Markers
subgenusEquisetum
subgenusHippochaete
FI G. 5. Lichenase digestion products of hemicellulose B from nine Equisetum species. The TLCs were developed either in butanol/ethanol/water (A) or inbutanol/acetic acid/water (B), and stained with thymol. A lichenase digest of the hemicellulose B from an MLG-rich grass, Festuca arundinacea, was run inparallel. Abbreviations: H, herbarium specimen; M, mature tissue; Y, young tissue; Glc, glucose; Lam2, laminaribiose; MLG3-4, tri- and tetrasacchariderepeat-unit of MLG; Mlt2-10, maltose to maltodecaose (‘malto-ladder’ obtained by partial hydrolysis of amylose on long-term storage of a sterile aqueous so-
lution). Spots marked ‘*’ are contamination, not seen on replicate plates.
Xue & Fry — Monilophyte hemicelluloses 881
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
DISCUSSION
Evolution of MLG in Equisetum
The fact that MLG is consistently abundant in the subgenusEquisetum, and scarce or very scarce in E. hyemale andE. myriochaetum but relatively high in E. ramosissimum,
suggests fluctuations in MLG biosynthesis during the evolu-tion of the horsetails. Likewise, in the Poales, levels of MLGcan vary dramatically between species, subspecies and var-ieties and even between genetically identical plants grownunder different environmental conditions (Stone and Clarke,1992).
FI G. 6. Lichenase digestion products of hemicellulose B from additional Equisetum species. Other details as in Fig. 5.
Xue & Fry — Monilophyte hemicelluloses882
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
Sel
agin
ella
will
deno
wii
YP
silo
tum
nud
um Y
Psi
lotu
m n
udum
M
E. a
rven
se Y
E. a
rven
se M
Euspor-angiate
Equisetum
Red: subgenus EquisetumBlue: subgenus Hippochaete
Eusporangiateferns
Leptosporangiateferns
Gymno-sperm
Basaldicot
E. f
luvi
atile
YE
. hye
mal
e Y
E. h
yem
ale
M
E. h
yem
ale
M
E. m
yrio
chae
tum
Y
E. m
yrio
chae
tum
ME
. pra
tens
e Y
E. p
rate
nse
ME
. x li
tora
le Y
E. x
lito
rale
ME
. x r
ober
tsii
Y
E. x
rob
erts
ii M
Mar
attia
frax
inea
Y
Mar
attia
frax
inea
MA
ngio
pter
is e
vect
a Y
Ang
iopt
eris
eve
cta
MA
ngio
pter
is li
godi
ifolia
YA
ngio
pter
is li
godi
ifolia
MO
smun
da r
egal
is Y
Osm
unda
reg
alis
MTo
dea
barb
ara
Y
Tode
a ba
rbar
a M
Tric
hom
anes
spe
cios
um M
Tric
hom
anes
spe
cios
um Y
Dip
teris
con
juga
ta Y
Dip
teris
con
juga
ta M
Mar
kers
Lygo
dium
japo
nicu
m Y
Lygo
dium
japo
nicu
m M
Lygo
dium
japo
nicu
m M
Ane
mia
sp.
YA
nem
ia s
p. M
Mar
sile
a dr
umm
ondi
i YM
arsi
lea
drum
mon
dii M
Thy
rsop
teris
ele
gans
YT
hyrs
opte
ris e
lega
ns M
Cya
thea
spi
nulo
sa Y
Cya
thea
spi
nulo
sa M
Woo
dsia
obt
usa
YW
oods
ia o
btus
a M
Cha
mae
cyp.
law
soni
ana
YC
ham
aecy
p. la
wso
nian
a M
Mar
kers
Nym
phae
a lo
tus
lam
ina
YN
ymph
aea
lotu
s pe
tiole
YN
ymph
aea
lotu
s pe
tiole
M
Mar
kers
Gal
GlcDP2DP3
DP6?DP7DP8DP9F
DP9NF
DP10
GalA
XXXG
XXLG
XLLG
Mar
kers
FI G. 7. Xyloglucan endoglucanase (XEG) digestion products of hemicellulose B from numerous monilophytes and three other vascular plants. The TLCs were developed in butanol/acetic acid/water, andstained with thymol. Markers (labelled at left): Gal, galactose; GalA, galacturonic acid; XXXG, XXLG and XLLG, oligosaccharides of DP 7, 8 and 9, respectively, from tamarind xyloglucan. Size-classes ofproducts (labelled at right) are indicated by their degree of polymerization (DP); e.g. DP7 is a heptasaccharide. DP9F, Nonasaccharides containing a fucose residue; DP9NF, lacking fucose. One residue offucose (6-deoxy-L-galactose) considerably increases chromatographic mobility compared with an oligosaccharide of similar size lacking a deoxy-sugar. The DP9F band is highlighted with a yellow stripe.
Abbreviations: M, mature tissue; Y, young tissue.
Xu
e&
Fry
—M
on
ilop
hyte
hem
icellulo
ses8
83
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
The phylogenetic position of E. bogotense is suggested to beeither sister to Hippochaete or basal to the entire genus (DesMarais et al., 2003; Guillon, 2004, 2007). Thus the abundancein E. bogotense of an MLG built almost only of the tetrasac-charide repeat-unit, G4G4G3G, suggests that this structure isa primordial feature of the genus Equisetum. In all otherEquisetum species, the disaccharide repeat-unit G3G is alsoan appreciable component. MLG has been retained abundantlyin the subgenus Equisetum, but almost lost in some membersof the subgenus Hippochaete. Its reappearance in oneHippochaete species (E. ramosissimum) was in a form witha highly unusual repeat-unit composition, predominantlybased on the disaccharide G3G. This may indicate that MLGacquired a subtly different role in this species. However,E. ramosissimum is closely related to E. hyemale (DesMarais et al., 2003; Guillon, 2004, 2007), implying a veryrapid evolution of MLG structure.
It has been suggested that the Equisetopsida total groupdiverged from the Marattiales in the period frommid-Triassic to Upper Palaeozoic (238–295 million yearsago) (Des Marais et al., 2003; Pryer et al., 2004). The com-plete absence of MLG in the extant Marattiales and all othernon-Equisetum monilophytes, and its presence in allEquisetum species, would suggest that MLG was acquiredlater than the Equisetopsida–Marattiales split and at least bythe crown group diversification in the Tertiary or Mesozoic.Nevertheless, it cannot be excluded that MLG was originallypresent in all eusporangiate monilophytes but then lostexcept in Equisetum. The invention of MLG in the seedplant lineage appears to be an entirely separate event. ThePoales date back to the Cretaceous (.65 million years ago)(Bremer, 2002) and poalean MLG is therefore assumed tohave been acquired around this time.
The significance of the different tetrasaccharide : trisacchar-ide : disaccharide ratios in the MLGs of different species is un-certain. An MLG composed mainly of one specific repeat-unit(either trisaccharide or tetrasaccharide) will tend to self-aggregate, thus readily coming out of aqueous solution orforming a stiff gel (Lazaridou et al., 2004). Such MLGs arethose of the lichen Cetraria islandica and many Equisetumspecies (with predominantly the trisaccharide or the tetrasac-charide repeat-unit, respectively). We have indeed noted thatpure dried MLG from Equisetum is difficult to redissolve inwater. Correspondingly, wheat MLG, which is mainly com-posed of the trisaccharide repeat-unit (trisaccharide : tetrasac-charide ratio ≈ 4.5 : 1), is less water-soluble than oat MLG,which has a less extreme ratio (trisaccharide :tetrasaccharide ≈ 2 : 1) (Lazaridou et al., 2004; Li et al., 2006).
It may be predicted that MLGs possessing a high proportionof disaccharide repeat-unit (e.g. that of E. ramosissimum) willbe highly flexible molecular chains, since the (1 � 3) bondallows free rotation. Where a disaccharide is flanked by twotetrasaccharides, there is a six-glucosyl run with alternating(1 � 3) and (1 � 4) bonds ( . . . G4G3G4G3G4G . . . ); suchdomains would be expected neither to self-aggregate readilynor to hydrogen-bond to the surface of cellulose microfibrils.The self-aggregation and solubility of MLGs and theirability to hydrogen-bond to cellulose are all features thatmay be expected to influence the biological characteristics ofthis cell-wall polysaccharide.
Evolution of xyloglucan in the monilophytes
Although small amounts of xyloglucan may be present inmature xylem walls (Mellerowicz et al., 2008), this hemicellu-lose is mainly a component of primary cell walls. As thereforeexpected, young tissues were richer in xyloglucan than maturetissues, which possess a higher proportion of secondary walls.Xyloglucan-derived oligosaccharides were detectable in allmonilophytes tested, though it was a very minor componentin some. In fact, xyloglucan is thought to be present in all land-plants; however, it appears not to be essential for viability inarabidopsis (Cavalier et al., 2008), suggesting that othercell-wall components can replace it functionally whenxyloglucan biosynthesis is compromised.
Among the eusporangiate monilophytes, the total xyloglu-can content showed interesting taxonomic trends. It wasscarce throughout the MLG-rich horsetail subgenusEquisetum, suggesting that MLG and xyloglucan may becapable of serving equivalent roles in primary cell-wall archi-tecture. It was also very low in abundance in Psilotum nudum,which is extremely rich in a different hemicellulose, gluco-mannan (Popper and Fry, 2004), suggesting that the lattermay be able to replace xyloglucan functionally. Indeed,mannose-rich polysaccharides are consistently abundant inthe cell walls of eusporangiate monilophytes (Popper andFry, 2004; Nothnagel and Nothnagel, 2007). On the otherhand, xyloglucan was relatively abundant in the MLG-poorhorsetail subgenus Hippochaete and in the (MLG-free) euspor-angiate ferns Marattia and Angiopteris. Most of the(MLG-free) leptosporangiate ferns also had high xyloglucancontents in their young tissues, although Dipteris, Lygodiumand Marsilea had less. It will be interesting to determinewhether these last three fern genera functionally replacexyloglucan with a different hemicellulose.
A ‘core pattern’ of five xyloglucan repeat-units,co-migrating on TLC with XLFG, XLLG, XXFG, XXLGand XXXG, was recognizable in all monilophytes studied, re-sembling that in the two representative seed-plants tested (agymnosperm and a basal dicot). This core pattern evenapplies to E. hyemale, which has recently been reported topossess several unusual xyloglucan repeat-units not foundin most other plants (Pena et al., 2008). It is likely that TLCin a single solvent system does not separate oligosaccharidescontaining the L repeat-unit from those in which L is replacedby the chemically similar D, nor those containing F from thosein which F is replaced by the chemically similar E (Fig. 2).Thus, the DP10 band in E. hyemale (and possibly otherEquisetum spp.; Fig. 7) is expected to include all three ofthe very similar fucose-containing decasaccharides reportedby Pena et al. (2008): XLEG, XLFG and XDEG. Likewise,the DP9F band will include both XXFG and XXEG; theDP9NF band XLDG, XLLG and XDDG; and the DP8 bandXXLG, XLXG and XXDG. If the E. hyemale oligosaccharidesreported by Pena et al. (2008) are grouped into the five classesthat are resolvable by TLC, their data show a DP10 : DP9NF :DP9F : DP8 : DP7 ratio of 23 : 46 : 5 : 22 : 4. In contrast, ourTLC patterns (which are similar for young E. hyemale andE. myriochaetum) show a ratio of roughly 24 : 7 : 17 : 23 : 29(estimated by intensity of thymol staining; Fig. 7). Althoughour data are only semi-quantitative, they nevertheless show
Xue & Fry — Monilophyte hemicelluloses884
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
appreciable differences from the ratios found by Pena et al.(2008), especially in exhibiting a higher abundance of the hep-tasaccharide and the fucose-containing nonasaccharides andmuch less of the non-fucosylated nonasaccharides such asXLLG.
Interspersed with the five ‘core pattern’ bands are additionalbands that show greater taxonomic diversity. For example,many of the species tested also possess a probable hexasac-charide. This may be XXGG, a major repeat-unit of manymembers of the Poales and Solanales (Hoffman et al., 2005;Fry, 2011), although it was not definitively identified. TheDP6 component was found in varying abundance across themonilophytes and in the basal early-diverging dicotNymphaea, but was least abundant in the genus Equisetum,supporting the view that hemicellulose structures show phylo-genetically based variation.
CONCLUSIONS
After confirming that MLG is confined, among all monilo-phytes tested, to Equisetum, we also show varied MLG struc-tures among the subgenera Equisetum and Hippochaete. Thesister group E. bogotense possesses its own potentially ances-tral MLG structure, very predominantly composed of the tetra-saccharide repeat-unit, G4G4G3G. Other members of thegenus seem to have refined this simple structure, e.g. varyingin the tetrasaccharide : disaccharide ratio. Furthermore, MLGwas lost as a predominant hemicellulose of the primary cellwall (by E. hyemale and E. ramosissimum) at least onceduring the adaptive radiation of the genus.
Xyloglucan, in contrast to MLG, is found in all land plantstested, and a ‘core pattern’ of repeat-units (co-migrating withXLFG, XXFG, XXLG/XLXG, XXXG) appears consistentlyin the TLC profiles of all species tested. However, xyloglucanhas become a very minor hemicellulose in some monilophytes –especially Psilotum and the horsetail subgenus Equisetum,which possess abundant alternative hemicelluloses (mannansin both; also MLG in Equisetum).
It is clear that land plants have experimented extensivelywith the structures and proportions of their hemicellulosesduring evolution.
ACKNOWLEDGEMENTS
We thank RBGE horticultural staff Fiona Inch and AndrewEnsoll for the collection of plant materials, and New YorkBotanic Garden for herbarium material of E. bogotense. Wethank Christopher Jeffree and Quentin Cronk for constructivecomments on the manuscript. This work was supported bythe BBSRC.
LITERATURE CITED
APG II: The Angiosperm Phyogeny Group. 2003. An update of theAngiosperm Phyologeny Group classification for the orders and familiesof flowering plants: APG II. Botanical Journal of the Linnean Society141: 399–436.
Baron R. 1889. Notes on the geology of Madagascar: with an appendix on thefossils. Quarterly Journal of the Geological Society 45: 305–331.
Bateman RM. 1991. Palaeobiological and phylogenetic implications ofanatomically-preserved Archaeocalamites from the Dinantian of Oxroad
Bay and Loch Humphrey Burn, southern Scotland. PalaeontographicaB 223: 1–59.
Bremer K. 2002. Gondwanan evolution of the grass alliance of families(Poales). Evolution 56: 1374–1387.
Buckeridge MS, Vergara CE, Carpita NC. 1999. The mechanism of synthe-sis of a mixed-linkage (1�3), (1�4)-b-D-glucan in maize: evidence formultiple sites of glucosyl transfer in the synthase complex. PlantPhysiology 120: 1105–1116.
Buckeridge MS, Rayon C, Urbanowicz B, Tine MAS, Carpita NC. 2004.Mixed linkage (1�3), (1�4)-b-D-glucans of grasses. CerealChemistry 81: 115–127.
Carpita NC, McCann MC. 2010. The maize mixed-linkage (1�3),(1�4)-b-D-glucan polysaccharide is synthesized at the Golgi membrane.Plant Physiology 153: 1362–1371.
Cavalier DM, Lerouxel O, Neumetzler L, et al. 2008. Disruption of twoArabidopsis thaliana xylosyltransferase genes results in plants deficientin xyloglucan, a major primary cell wall component. The Plant Cell20: 1519–1537.
Channing A, Zamuner A, Edwards D, Guido D. 2011. Equisetum thermalesp. nov. (Equisetales) from the Jurassic San Agustın hot spring deposit,Patagonia: anatomy, paleoecology, and inferred paleoecophysiology.American Journal of Botany 98: 680–697.
Delevoryas T. 1962. Morphology and evolution of fossil plants. New York,NY: Holt, Rinehart and Winston.
Des Marais DL, Smith AR, Britton DM, Pryer KM. 2003. Phylogeneticrelationships and evolution of extant horsetails, Equisetum, based onchloroplast DNA sequence data (rbcl and trnl-f ). International Journalof Plant Sciences 164: 737–751.
Eder M, Tenhaken R, Driouich A, Lutz-Meindl U. 2008. Occurrence andcharacterization of arabinogalactan-like proteins and hemicelluloses inMicrasterias (Streptophyta). Journal of Phycology 44: 1221–1234.
Ford CW, Percival E. 1965. The carbohydrates of Phaeodactylum tricornu-tum. Part i. Preliminary examination of the organism, and characterisationof low molecular weight material and of a glucan. Journal of theChemical Society 1965: 7035–7041.
Frankova L, Fry SC. 2011. Phylogenetic variation in glycosidases and glyca-nases acting on plant cell wall polysaccharides, and the detection of trans-glycosidase and trans-b-xylanase activities. The Plant Journal 67:662–681.
Fry SC. 2000. The growing plant cell wall: chemical and metabolic analysis,Reprint edn. Caldwell, NJ: The Blackburn Press.
Fry SC. 2011. Cell wall polysaccharide composition and covalent crosslink-ing. Annual plant reviews: plant polysaccharides, biosynthesis and bio-engineering, Vol. 41. Ulvskov P. ed. Oxford: Blackwell Publishing .
Fry SC, York WS, Albersheim P, et al. 1993. An unambiguous nomenclaturefor xyloglucan-derived oligosaccharides. Physiologia Plantarum 89: 1–3.
Fry SC, Mohler KE, Nesselrode BHWA, Frankova L. 2008a.Mixed-linkage b-glucan: xyloglucan endotransglucosylase, a novel wall-remodelling enzyme from Equisetum (horsetails) and charophytic algae.The Plant Journal 55: 240–252.
Fry SC, Nesselrode BHWA, Miller JG, Mewburn BR. 2008b.Mixed-linkage (1�3,1�4)-b-D-glucan is a major hemicellulose ofEquisetum (horsetail) cell walls. New Phytologist 179: 104–115.
Graham LE, Wilcox L. 1999. Algae. San Francisco, CA: BenjaminCummings.
Guillon JM. 2004. Phylogeny of horsetails (Equisetum) based on the chloro-plast rps4 gene and adjacent noncoding sequences. Systematic Botany 29:251–259.
Guillon JM. 2007. Molecular phylogeny of horsetails (Equisetum) includingchloroplast atpb sequences. Journal of Plant Research 120: 569–574.
Harris PJ. 2005. Diversity in plant cell walls. In: Henry RJ. ed. Plant diversityand evolution: genotypic and phenotypic variation in higher plants.Wallingford, UK: CAB International, 201–227.
Hauke RL. 1959. A taxonomic monograph of the genus Equisetum subgenusHippochaete. PhD Thesis, University of Michigan, Ann Arbor, MI.
Hoffman M, Jia Z, Pena MJ, et al. 2005. Structural analysis of xyloglucansin the primary cell walls of plants in the subclass Asteridae. CarbohydrateResearch 340: 1826–1840.
Xue & Fry — Monilophyte hemicelluloses 885
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
ecember 2021
Kenrick P, Crane PR. 1997a. The origin and early evolution of plants onland. Nature 389: 33–39.
Kenrick P, Crane PR. 1997b. The origin and early diversification of landplants: a cladistic study. Smithsonian Series in ComparativeEvolutionary Biology. Washington, DC: Smithsonian Institution Press.
Lazaridou A, Biliaderis CG. 2007. Cryogelation phenomena in mixed skimmilk powder–barley b-glucan–polyol aqueous dispersions. FoodResearch International 40: 793–802.
Lazaridou A, Biliaderis CG, Micha-Screttas M, Steele BR. 2004. A com-parative study on structure–function relations of mixed-linkage (1�3),(1�4) linear b-D-glucans. Food Hydrocolloids 18: 837–855.
Li W, Cui SW, Kakuda Y. 2006. Extraction, fractionation, structural andphysical characterization of wheat b-D-glucans. Carbohydrate Polymers63: 408–416.
Ligrone R, Vaughn KC, Renzaglia KS, Knox JP, Duckett JG. 2002.Diversity in the distribution of polysaccharide and glycoprotein epitopesin the cell walls of bryophytes: new evidence for the multiple evolution ofwater-conducting cells. New Phytologist 156: 491–508.
Luttenegger DG, Nevins DJ. 1985. Transient nature of a (1�3),(1�4)-b-glucan in Zea mays coleoptile cell walls. Plant Physiology77: 175–178.
Matsunaga T, Ishii T, Matsumoto S, et al. 2004. Occurrence of the primarycell wall polysaccharide rhamnogalacturonan II in pteridophytes, lyco-phytes, and bryophytes: implications for the evolution of vascularplants. Plant Physiology 134: 339–351.
Meikle PJ, Hoogenraad NJ, Bonig I, Clarke AE, Stone BA. 1994. A(1�3,1�4)-b-glucan-specific monoclonal antibody and its use in thequantitation and immunocytochemical location of (1�3,1�4)-b-glucans. The Plant Journal 5: 1–9.
Mellerowicz EJ, Immerzeel P, Hayashi T. 2008. Xyloglucan: the molecularmuscle of trees. Annals of Botany 102: 659–665.
Nevo Z, Sharon N. 1969. Cell wall of Peridinium westii: a non cellulosicglucan. Biochimica et Biophysica Acta 173: 161–175.
Nothnagel AL, Nothnagel EA. 2007. Primary cell wall structure in the evo-lution of land plants. Journal of Integrative Plant Biology 49:1271–1278.
Pauly M, Anderson LN, Kaupinnen S, Kofod LV, Darvill AG. 1999. Axyloglucan specific endo-b-1,4-glucanase from Aspergillus aculeatus: ex-pression in yeast, purification and characterisation of the recombinantenzyme. Glycobiology 9: 93–100.
Pena MJ, Darvill AG, Eberhard S, York WS, O’Neill MA. 2008. Moss andliverwort xyloglucans contain galacturonic acid and are structurally dis-tinct from the xyloglucans synthesized by hornworts and vascularplants. Glycobiology 18: 891–904.
Popper ZA. 2008. Evolution and diversity of green plant cell walls. CurrentOpinion in Plant Biology 11: 286–292.
Popper ZA, Fry SC. 2003. Primary cell wall composition of bryophytes andcharophytes. Annals of Botany 91: 1–12.
Popper ZA, Fry SC. 2004. Primary cell wall composition of pteridophytesand spermatophytes. New Phytologist 164: 165–174.
Pryer KM, Schneider H, Smith AR, et al. 2001. Horsetails and ferns are amonophyletic group and the closest living relatives to seed plants.Nature 409: 618–622.
Pryer KM, Schuettpelz E, Wolf PG, Schneider H, Smith AR, Cranfill R.2004. Phylogeny and evolution of ferns (monilophytes) with a focus onthe early leptosporangiate divergences. American Journal of Botany 91:1582–1598.
Smith BG, Harris PJ. 1999. The polysaccharide composition of Poales cellwalls: Poaceae cell walls are not unique. Biochemical Systematics andEcology 27: 33–53.
Sørensen I, Pettolino FA, Wilson SM, et al. 2008. Mixed-linkage(1�3,1�4)-b-D-glucan is not unique to the Poales and is an abundantcomponent of Equisetum arvense cell walls. The Plant Journal 54:510–521.
Sørensen I, Domozych D, Willats WGT. 2010. How have plant cell wallsevolved? Plant Physiology 153: 366–372.
Stace CA. 1981. Plant taxonomy and biosystematics, 2nd edn. Cambridge:Cambridge University Press.
Stanich NA, Rothwell GW, Stockey RA. 2009. Phylogenetic diversificationof Equisetum (Equisetales) as inferred from lower cretaceous speciesof British Columbia, Canada. American Journal of Botany 96:1289–1299.
Stebbins GL. 1992. Comparative aspects of plant morphogenesis: a cellular,molecular and evolutionary approach. American Journal of Botany 79:589–598.
Stone BA, Clarke AE. 1992. Chemistry and biology of (1�3)-b-glucans.Bundoora, Victoria, Australia: La Trobe University Press.
Trethewey JAK, Campbell LM, Harris PJ. 2005. (1�3),(1�4)-b-D-glucans in the cell walls of the Poales (sensu lato): an immu-nogold labeling study using a monoclonal antibody. American Journal ofBotany 92: 1660–1674.
Woodward JR, Fincher GB, Stone BA. 1983. Water-soluble (1�3),(1�4)-b-D-glucans from barley (Hordeum vulgare) endosperm. II.Fine structure. Carbohydrate Polymers 3: 207–225.
Xue & Fry — Monilophyte hemicelluloses886
Dow
nloaded from https://academ
ic.oup.com/aob/article/109/5/873/141003 by guest on 28 D
