Matrix polysaccharides in primary and secondary plant cell walls are biochemically diverse and include xylans, glucomannans, β-glucans (i.e., mixed linkage β-glucan and xyloglucan), pectins, and β-galactan. Their composition and molecular structure in specific cell walls depend on the type of plant, type of tissue, and the temporal development of the plant. The supramolecular organization of matrix polysaccharides around the cellulose microfibrils affects the flexibility and strength of the cell wall. However, the molecular level details of the interface between the cellulose microfibrils and the matrix polysaccharides are not fully understood. Here, the interaction of unsubstituted model oligosaccharides with cellulose microfibrils was investigated through molecular dynamics simulations of the adsorption of model oligosaccharides representing their respective backbone motifs. The simulations show that induced conformational changes of the polysaccharide backbone upon adsorption and its alignment with the cellulose microfibril lead to stronger interactions with cellulose. This differentiates typical primary and secondary cell wall hemicelluloses (xylans, glucomannans, and β-glucans) from pectins and β-galactan and explains why mixed-linkage β-glucan can be classified as a hemicellulose. Our study contributes to the development of accurate molecular models for plant cell walls, which will improve our understanding of lignocellulosic biomass and its conversion into functional biobased materials.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

Glucuronoxylan is the main hemicellulose in the secondary cell wall of angiosperms. Elucidating its molecular structure provides a basis for more accurate plant cell wall models and the utilization of xylan in biorefinery processes. Here, we investigated the spacing of acetyl, glucuronopyranosyl and galactopyranosyl substitutions on Eucalyptus glucuronoxylan using sequential extraction combined with enzymatic hydrolysis and mass spectrometry. We found that the acetyl groups are preferentially spaced with an even pattern and that consecutive acetylation is present as a minor motif. Distinct odd and even patterns of glucuronidation with tight and sparse spacing were observed. Furthermore, the occurrence of consecutive glucuronidation is reported, which adds to the growing body of evidence that this motif is not only present in gymnosperms but also in angiosperms. In addition, the presence of terminal galactopyranosyl units, which can be released by β-galactosidase, altered the digestibility of the glucuronoxylan by GH30 and GH10 xylanase and appeared to be clustered within the polymeric backbone. These findings increase our understanding of the complex structure of glucuronoxylans and its effect on the extractability and biological degradation of Eucalyptus wood.

The properties of wood cell walls are determined by the composition and the molecular structures of the cell wall polymers (cellulose, hemicelluloses, pectins and lignin) and the interactions between them. In particular, hemicelluloses are an underutilized source of biopolymers that constitute around 30 % of wood. In hardwoods, the main hemicellulose is acetylated glucuronoxylan and the variation in the patterns of acetylation and glucuronidation between tree species is not fully understood. This study aimed to increase understanding of the xylan structure in particular and more generally, the interaction between the cell wall matrix polysaccharides and cellulose, which are important for the preparation of more accurate cell wall models as well as for the development of hemicellulose-based products.

The effect of matrix polysaccharides’ backbone on the assembly with cellulose fibril in water was studied using atomistic simulations. First, several setups were compared with xylo-oligosaccharides (XOs) as model hemicellulose motifs. Anti-parallel alignment together with the conformational change to a 2-fold helix as well as the formation of a distinct hydrogen bonding network were characteristics of spontaneous adsorption of XOs to cellulose. The established simulation setup was further used to expand the scope to other hemicelluloses and pectins. The parameters investigated included mobility on the cellulose surface, alignment, conformation in water and on cellulose and the interaction strength. Alignment along the fibril and conformational change upon adsorption defined the adaptability of hemicelluloses and distinguished them from the other oligosaccharides. In this sense, the mixed-linked β-glucan with a β-(1→3)-linkage was shown to be comparable to β-(1→4)-linked hemicelluloses.

To investigate the structure of hemicelluloses, sequential subcritical water (SWE) and alkaline extraction methods were applied to aspen and Eucalyptus wood. Buffered SWE released acetylated glucuronoxylan (acGX) with distinct patterns of acetylation and glucuronidation and enabled the extraction of galactosylated acGX from Eucalyptus without the need for prior delignification. In particular, a small amount of consecutive glucuronidation in both Eucalyptus and aspen xylan showed a more complex substitution pattern than previously thought, with similarity to arabinoglucuronoxylan from softwoods. The structure of GX affected its biological degradability by xylanases. Regions of low degree of glucuronidation were shown to aggregate in water, which hindered their accessibility to GH30 glucuronoxylanase. This was particularly relevant to alkali-extracted GX devoid of acetylation. In Eucalyptus, galactosylation impeded the digestion by GH30. GH10 could release galactosylated XOs but the cleavage site preference was shown to be altered. The large difference in the amount of Gal-MeGlcA from incubations with GH30 and GH10 suggests that the Gal-MeGlcA motifs may be clustered in xylan. Furthermore, the terminal galactose was released by a β- but not an α-galactose confirming the presence of galactose as a β-anomer.

The degree and the pattern of glucuronidation were further shown to affect the aggregation of glucuronoxylan at acidic pH 2.0. Separation of beech glucuronoxylan in two fractions based on their solubility at pH 2.0 was achieved by freezing and subsequent thawing. The precipitated GX had a higher DP and a lower degree of glucuronidation with an enrichment in the motifs with an even and lengthily-spaced glucuronidation pattern (X>6U) compared to the soluble GX. The fractionation of these GX populations in acidic conditions revealed the occurrence of interpolymer variability of glucuronidation, possibly corresponding to different activities of the two known glucuronyltransferases GUX1 and GUX2.

Overall, these results provide insights into the fundamentals of the molecular structure of xylans and the interaction of matrix polysaccharides with cellulose fibrils, which can increase our understanding of their biological functions and be utilized in the development of more accurate cell wall models and hemicellulose-based products.

Träcellväggens egenskaper bestäms av sammansättningen och den molekylära strukturen hos cellväggens polymerer (cellulosa, hemicellulosa, pektin och lignin) samt deras växelverkningar. I synnerhet hemicellulosa som utgör omkring 30% av trä är en underutnyttjad källa till biopolymerer. Acetylerad glukoronoxylan utgör den huvudsakliga hemicellulosan i lövträ men anledningen till variationen i glukuronisering och acetylering som existerar mellan olika träslag är inte helt förstådd.

Målet för arbetet som presenteras här var ökad kunskap om xylanstruktur och, mer allmänt, växelverkningar mellan de oordnade polysackariderna i cellväggen och cellulosa. Dessa är viktiga för att konstruera korrekta cellväggsmodeller samt för att utveckla produkter baserade på hemicellulosa. Inledningsvis undersöktes effekten av hemicellulosornas huvudkedjor på hur de ansamlas på cellulosafibriller med hjälp av atomistiska datorsimuleringar. Flera system där xylo-oligosackarider (XO) användes som modeller för hemicellulosastruktur jämfördes. Den spontana adsorptionen av XO till cellulosa som observerades ledde till anti-parallell upplinjering, en konformationsändring till en tvåfaldig helix samt bildandet av regelbundna nätverk av vätebindningar. Dessa modeller utökades senare till att innefatta andra hemicellulosor samt pektin. Parametrarna som undersöktes var mobilitet på cellulosaytor, upplinjering, konformation i lösning och på cellulosaytor samt styrkan hos växelverkningarna. Tydlig upplinjering samt en distinkt konformationsförändring till följd av adsorptionen var utmärkande för hemicellulosornas anpassningsbarhet vilket särskilde dem från de andra polysackariderna. Sett ur detta perspektiv var β-glukaner som inkluderar β-(1→3)-bindningar jämförbara med β-(1→4)-bundna hemicellulosor.

Sekventiell extraktion i subkritiskt vatten (SWE) och alkali utfördes på asprespektive eukalyptusträ med syftet att undersöka hemicellulosornas struktur. Buffrad SWE ledde till att acetylerad glukuronoxylan (acGX) med distinkta acetylerings- och glukuroniseringsmönster frisattes. Detta möjliggjorde extraktion av galaktosylerad acGX från eukalyptus utan att delignifiering behövdes. Värt att notera är att en liten andel glukuronsyror i följd, i både asp och eukalyptus, indikerar ett mer komplext substitutionsmönster än vad man tidigare trott, likt de i arabinoglukuronoxylan från barrträ. Strukturen i GX påverkade nedbrytbarheten genom xylanaser. Områden med låg glukuroniseringsgrad aggregerade i vatten, vilket minskade åtkomligheten för glukuronoxylanas GH30. Detta var särskilt relevant för alkaliextraherad GX vilken saknar acetylering. Vidare så hindrade galaktosideringen i eukalyptus åtkomligheten för GH30. GH10 kunde frisätta galaktosylerade XO, men med en förändrad preferens för vilken bindning den bryter. Stor skillnad i mängden Gal-MeGlcA från odlingar med GH30 och GH10 kan betyda att Gal-MeGlcA uppträder i kluster längs xylankedjan. Vidare så frisattes den terminala galaktosen som en β- och inte en α-galaktos, vilket bekräftar förekomsten av galaktos som dess β-anomer.

Substitutionsgrad och -struktur visades vidare påverka aggregering av GX vid surt pH 2.0. Genom frysning och efterföljande upptining kunde separering av GX från bok i två fraktioner uppnås, baserade på deras respektive löslighet vid pH 2.0. Den utfällda fraktionen hade högre polymerisationsgrad och lägre substitutionsgrad av glukoronsyra, men högre andel motiv där substituenterna var jämt fördelade med långa avstånd (X>6U) jämfört med den lösliga fraktionen. Fraktionering vid sura betingelser visade på förekomsten av variationer i glukuronisering mellan individuella polymerer vilket möjligen kan knytas till skillnaden i aktivitet mellan de två kända glukuronyltransferaserna GUX1 och GUX2.

Sammantaget ger resultaten insikter i grundläggande frågeställningar runt xylanernas molekylära struktur och växelverkningar mellan cellväggens olika oordnade polysackarider och cellulosafibriller. Detta kan öka vår kunskap om deras biologiska funktion samt användas för att utveckla mer detaljerade cellväggsmodeller och produkter baserade på hemicellulosa.

Puun soluseinien ominaisuudet määräytyvät soluseinäpolymeerien (selluloosa, hemiselluloosat, pektiinit ja ligniini) molekyylirakenteen sekä niiden välisten vuorovaikutusten perusteella. Vaikka 30% puun kuivapainosta muodostuu hemiselluloosista, niitä on hyödynnetty varsin vähän. Kaikkien lehtipuiden pääasiallinen hemiselluloosa on O-asetyyli-4-Ometyyliglukurono-β-D-ksylaani (acGX). Ei kuitenkaan ole vielä tarkasti tiedossa, kuinka paljon O-asetyyli ja MeGlcA ryhmien jakautuminen ptkin ksylaaniketjua vaihtelee puulajeittain. Tämän tutkimuksen tarkoitus onkin yhtäältä lisätä tietoa ksylaanin molekyylirakenteesta lehtipuissa ja toisaalta selvittää yleisemmin soluseinän polysakkaridien ja selluloosan vuorovaikutusta.

Tutkimuksen ensimmäisessä osassa selvitimme simulaatiokokeiden avulla kuinka polysakkaridiketjun molekyylirakenne (ilman sivuryhmiä) vaikuttaa niiden adsorboitumiseen selluloosan pinnalle. Simulaatiasetelmaa testattiin ensin ksylaanilla, jolloin havaittiin, että vapaasti adsorboituva ksylaani asettuu yleensä vastakkaiseen suuntaan selluloosan glukoosiketjuihin nähden, muuttaa konformaation 2-kierteiseksi sekä muodostaa vetysidosten verkoston selluloosan kanssa. Testattua simulaatiasetelmaa käytettiin edelleen muihin hemiselluloosiin ja pektiineihin. Tutkittuja parametreja olivat liikkuvuus selluloosan pinnalla, suuntautuminen, konformaatio vedessä ja selluloosan pinnalla sekä vuorovaikutuksen vahvuus. Tulokset osoittivat, että hemiselluloosat erosivat muista polysakkarideista konformaation ja suuntautumisen perusteella. Yllättävää oli, että myös β-glukaani, huolimatta yhdestä β-(1→3)-sidoksesta, käyttäytyi samalla tavalla kuin tyypilliset hemiselluloosat, jotka koostuvat β-(1→4)-sidoksin ketjuttuneista monosakkarideista.

Toisessa osassa selvitimme kokeellisin menetelmin ksylaanin molekyylirakennetta. Puskuroidun subkriittisen vesi- (SWE) ja alkaliuuton jaksottamisella saatiin glukuronoksylaania (GX) uutettua tehokkaasti haavasta ja eukalyptuksesta ilman erillistä esikäsittelyä ligniinin poistamiseksi. Puskuroitu SWE oli erityisen hyödyllinen asetyyli- (haapa ja eukalyptus) ja galaktoosiryhmien (eukalyptus) uutossa. Peräkkäisiä glukuronidiryhmiä havaittiin molempien puulajien ksylaanissa osoittaen, että sivuryhmät jakautuvat ksylaaniketjulle monimutkaisemmin kuin on tähän mennessä tiedetty. Sama substituutiokuvio on aiemmin havaittu havupuiden arabinoglukuronoksylaanissa. Lisäksi huomattiin, että ksylaanin rakenne vaikutti sen biologiseen hajoavuuteen ksylanaasien avulla. Pieni glukuronidaatioaste aiheutti ksylaanin yhteenkerääntymistä vedessä, mikä vaikeutti glukuronoksylanaasin toimintaa, erityisesti alkaliuutolla kerätyllä ksylaanilla, jossa ei ole asetyyliryhmiä parantamassa liukoisuutta. Eukalyptuksen ksylaanin galaktoosiryhmät estivät GH30 glukuronoksylanaasin toiminnan. GH10-ksylanaasin spesifisyys muuttui hieman eukalyptuksen galaktoosin vaikutuksesta. Suuri ero Gal-MeGlcA sivuryhmien määrässä GH30 ja GH10 inkubaatioista saaduissa ksylo-oligosakkarideissa viittasi siihen, että Gal-MeGlcA saattavat olla ryhmittyneinä tiettyyn osaan ksylaania. Galaktoosin hydrolysoituminen β-, muttei α-galaktanaasin avulla vahvisti galaktoosin esiintymisen β-anomeerina.

Glukuronidaation määrän ja laadun osoitettiin edelleen vaikuttavan glukuronoksylaanin saostumiseen happamissa olosuhteissa. Pyökin GX pystyttiin erottamaan kahteen fraktioon liukoisuuden perusteella, kun hapan liuos (pH 2.0) ensin jäädytettiin ja sitten sulatettiin. Saostuneella ksylaanilla oli korkeampi polymeroitumis-, matalampi glukuronidaatioaste ja tasaisemmin jakautuneet glukuronidiryhmät verrattuna liukoiseen ksylaaniin. Tämä osoitti, että substituutiokuvio vaihtelee sekä yksittäisen ksylaanipolymerin varrella että useiden ksylaanien välillä, mikä todennäköisesti on seurausta kahden tunnetun glukuronylitransferaasin, GUX1 ja GUX2 eri aktiivisuudesta.

Tutkimus antaa uutta tietoa ksylaanien molekyylirakenteesta ja matriisipolysakkaridien vuorovaikutuksen perusteista selluloosafibrillien pinnalla. Tietoa voidaan hyödyntää sekä tarkempien soluseinämallien että hemiselluloosapohjaisten tuotteiden kehityksessä.

Xylan is a fundamental structural polysaccharide in plant secondary cell walls and a valuable resource for biorefinery applications. Deciphering the molecular motifs of xylans that mediate their interaction with cellulose and lignin is fundamental to understand the structural integrity of plant cell walls and to design lignocellulosic materials. In the present study, we investigated the pattern of acetylation and glucuronidation substitution in hardwood glucuronoxylan (GX) extracted from aspen wood using subcritical water and alkaline conditions. Enzymatic digestions of GX with β-xylanases from glycosyl hydrolase (GH) families GH10, GH11 and GH30 generated xylo-oligosaccharides with controlled structures amenable for mass spectrometric glycan sequencing. We identified the occurrence of intramolecular motifs in aspen GX with block repeats of even glucuronidation (every 2 xylose units) and consecutive glucuronidation, which are unique features for hardwood xylans. The acetylation pattern of aspen GX shows major domains with evenly-spaced decorations, together with minor stretches of highly acetylated domains. These heterogenous patterns of GX can be correlated with its extractability and with its potential interaction with lignin and cellulose. Our study provides new insights into the molecular structure of xylan in hardwood species, which has fundamental implications for overcoming lignocellulose recalcitrance during biochemical conversion.

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharification and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cellulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreatment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries.

Interaction between xylan and cellulose microfibrils is required to maintain the integrity of secondary cell walls. However, the mechanisms governing their assembly and the effects on cellulose surface polymers are not fully clear. Here, molecular dynamics simulations are used to study xylan adsorption onto hydrated cellulose fibrils. Based on multiple spontaneous adsorption simulations it is shown that an antiparallel orientation is thermodynamically preferred over a parallel one, and that adsorption is accompanied by the formation of regular but orientation-dependent hydrogen bond patterns. Furthermore, xylan adsorption restricts the local dynamics of the adjacent glucose residues in the surface layer to a level of the crystalline core, which is manifested as a three-fold increase in their 13C NMR T1 relaxation time. These results suggest that xylan forms a rigid and ordered layer around the cellulose fibril that functions as a transition phase to more flexible and disordered polysaccharide and lignin domains.