The Building Blocks of Early Land Plants: Glycosyltransferases and Cell Wall Architecture in the model liverwort Marchantia polymorpha

The Building Blocks of Early Land Plants: Glycosyltransferases and Cell Wall Architecture in the model liverwort Marchantia polymorpha | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The Building Blocks of Early Land Plants: Glycosyltransferases and Cell Wall Architecture in the model liverwort Marchantia polymorpha View ORCID Profile H.S. Kang , X. Tong , A. Mariette , M. Leong , C. Beahan , View ORCID Profile E. Flores-Sandoval , G. B. Pedersen , C. Rautengarten , View ORCID Profile J.L. Bowman , B. Ebert , View ORCID Profile A. Bacic , View ORCID Profile M. Doblin , View ORCID Profile S. Persson , View ORCID Profile E.R. Lampugnani doi: https://doi.org/10.1101/2025.04.30.651426 H.S. Kang 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 2 Faculty of Biology and Biotechnology, Ruhr University Bochum , Bochum 44810, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for H.S. Kang X. Tong 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site A. Mariette 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 2 Faculty of Biology and Biotechnology, Ruhr University Bochum , Bochum 44810, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site M. Leong 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site C. Beahan 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site E. Flores-Sandoval 3 School of Biological Sciences, Monash University , Melbourne, VIC 3800 Australia 4 ARC Centre of Excellence for Plant Success in Nature and Agriculture, Monash University , Melbourne, VIC 3800 Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for E. Flores-Sandoval G. B. Pedersen 6 Department of Plant & Environmental Science, Copenhagen Plant Science Center (CPSC), University of Copenhagen , 1871 Frederiksberg, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site C. Rautengarten 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 2 Faculty of Biology and Biotechnology, Ruhr University Bochum , Bochum 44810, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site J.L. Bowman 3 School of Biological Sciences, Monash University , Melbourne, VIC 3800 Australia 4 ARC Centre of Excellence for Plant Success in Nature and Agriculture, Monash University , Melbourne, VIC 3800 Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for J.L. Bowman B. Ebert 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 2 Faculty of Biology and Biotechnology, Ruhr University Bochum , Bochum 44810, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site A. Bacic 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 5 La Trobe Institute for Sustainable Agriculture & Food, School of Agriculture , Biomedicine and Environment, La Trobe University , Bundoora, Victoria 3086, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for A. Bacic M. Doblin 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 5 La Trobe Institute for Sustainable Agriculture & Food, School of Agriculture , Biomedicine and Environment, La Trobe University , Bundoora, Victoria 3086, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for M. Doblin S. Persson 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 6 Department of Plant & Environmental Science, Copenhagen Plant Science Center (CPSC), University of Copenhagen , 1871 Frederiksberg, Denmark 7 Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Minhang 200240, Shanghai, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for S. Persson For correspondence: edwin.lampugnani{at}unimelb.edu.au E.R. Lampugnani 1 School of Biosciences, The University of Melbourne , Melbourne, VIC 3010, Australia 8 Menzies Institute for Medical Research, College of Health and Medicine, University of Tasmania , Hobart, TAS, 7000 Australia 9 School of Health Sciences, University of Melbourne , Parkville, VIC, 3010 Australia 10 Department of Medicine (RMH), Melbourne Medical School, University of Melbourne , Parkville, VIC, 3010 Australia 11 AirHealth Pty Ltd. , Brunswick, VIC, 3056 Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for E.R. Lampugnani For correspondence: edwin.lampugnani{at}unimelb.edu.au Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary The liverwort Marchantia polymorpha has emerged as an important plant model for developmental studies and may become central to elucidate the complex process of cell wall polysaccharide biosynthesis. This study comprehensively analyses the composition and structure of cell wall glycans across eight different M. polymorpha tissue types. We show that while the cell walls largely mirror known land plant cell wall composition, they also exhibit some unique characteristics. For example, α-(1,5)-arabinan was prominently present in the sporophyte tissue, which may indicate a specialised role in this life stage. Furthermore, M. polymorpha cell walls displayed a remarkably low overall pectin content, yet the abundance of pectic arabinan in sporophytes hint at its putative role in the evolution and complexity of spermatophyte cell walls. Through comparative analyses of glycosyltransferase (GT) families across plant species, we found that M. polymorpha has a diversified GT repertoire compared for example, to the model plant Arabidopsis thaliana , indicating uniqueness in its cell wall biosynthesis pathways. To support research underpinning cell wall biosynthesis, we developed a Gateway compatible compendium of 93 M. polymorpha GTs, providing a valuable resource for genetic and functional studies. Our study thus works as a foundation to drive new insights into cell wall evolution, structure and function across the plant kingdom. Introduction The plant cell wall is an extracellular matrix surrounding the plasma membrane which governs cell growth, and therefore the morphogenesis of plants ( Cosgrove, 2016 ). As the outer-most layer of the cell, the cell wall is an important part of plant immunity and resistance to (a)biotic stresses ( Bacete et al., 2018 ; Houston et al., 2016 ; Molina et al., 2021 ; Tenhaken, 2015 ). The plant cell wall is the most abundant source of biomass on Earth and important as a food, fibre and fuel ( Bar-On et al., 2018 ), and its properties confer many anthropogenically important properties, such as the strength of timber for building materials and as a source of dietary fibre that is essential for gut health ( Johnson et al., 2018 ). Due to its utility, it is of great interest to understand how plant cell wall composition determined at a molecular level. The plant primary cell wall is composed mostly of polysaccharides and some glycosylated proteins (glycoproteins). Cell wall polysaccharides are further subdivided into three major groups – cellulose, hemicelluloses, and pectins ( Lampugnani et al., 2018 ). Cellulose is a 1,4-β-linked glucose (Glc) homopolymer where 18 glucan chains biosynthesised in parallel form microfibrils, which are the main load bearing structure of the plant cell wall ( McFarlane et al., 2014 ). Hemicelluloses are a diverse group of polysaccharides including xyloglucans, heteromannans and heteroxylans ( Scheller & Ulvskov, 2010 ). They are characterised by the equatorial 1,4-β-linked backbone of Glc, mannose (Man) and xylose (Xyl) respectively, and are substituted further by other sugars and non-glycosyl substituents. Pectins, the other major polysaccharide group, form the hydrated gel-like matrix of the cell wall and are also very important for cell-cell adhesion facilitated by the middle lamella ( Atmodjo et al., 2013 ; Mohnen, 2008 ). Pectins are also the most heterogeneous plant cell wall polysaccharides, and consist of homogalacturonan, rhamnogalacturonan-I and -II (RG-I and RG-II). Lastly, glycoproteins comprise approx. 10% (w/w) in the primary cell wall and include arabinogalactan proteins (AGPs) and extensins, which are part of the hydroxyproline-rich glycoprotein (HRGPs) superfamily ( Nguema-Ona et al., 2014 ; Strasser et al., 2021 ). While most cell wall polymers associate through non-covalent interactions (both hydrogen bonds and ionic complexes (e.g. pectins through Ca 2+ )) some components can be covalently attached to one another ( Tan et al., 2013 , 2022 ; Doblin et al., 2022). Despite this overarching understanding of the composition of the cell wall, there are still large gaps in our knowledge about how cell wall polysaccharides and glycoproteins are assembled, and which biosynthetic enzymes and auxiliary proteins underpin their biosynthesis. In part, this is because of the complex network of genes required for the biosynthesis of the many components of the wall. To illustrate, it is estimated that about 15% of genes contribute to the synthesis and remodelling of cell walls in Arabidopsis (Carpita et al. 2001), and it is estimated that pectins alone require at least 67 different types of transferase enzymes for their biosynthesis ( Mohnen, 2008 ). Additionally, apart from cellulose, the other classes of polysaccharides display cell/tissue-type, developmental and taxonomic variability ( Doblin et al., 2023 ). For example, xyloglucans in land plants show lineage specific sidechain substitutions ( Peña et al., 2008 ). The heteromannans are further divided into glucomannans, galactomannans and galactoglucomannan depending on their backbone and sidechains ( Voiniciuc, 2022 ). Heteroxylan sub-classes include arabinoxylans, glucuronoxylans and glucouronoarabinoxylans ( Curry et al., 2023 ). Other important cell wall polysaccharides include callose ( Doblin et al., 2023 ; B. Wang et al., 2022 ), mixed-linkage β-glucans (MLG) ( Doblin et al., 2023 ; S. J. Kim & Brandizzi, 2016 ) and arabinoglucan ( Roberts et al., 2018 ). Furthermore, hemicelluloses and pectins are commonly substituted with non-glycosyl residues (e.g O -acetyl, O -methyl, phenolic acids (e.g. ferulic acid)) adding further complexity to the assembly process. The high degree of structural complexity of plant cell wall polysaccharides indicates an equally complex mechanism of biosynthesis. Plant cell wall polysaccharides are synthesised by glycosyltransferases (GTs), enzymes that catalyse glycosidic bonds between an activated sugar donor and specific acceptors such as proteins, lipids and other sugars ( Lairson et al., 2008 ; Moremen & Haltiwanger, 2019 ). GTs are catalogued in an online database called CAZy (Carbohydrate Active Enzymes; https://www.cazy.org/ ), which currently contains over 1 400 000 GTs in more than 130 families ( Drula et al., 2022 ). Plant cell wall biosynthetic GTs belong to 18 well-defined clades which represent subsets of the larger GT superfamily ( Table 1 ). Within these clades, the eudicot model plant Arabidopsis thaliana has 288 GT genes. In total, Arabidopsis encodes 553 GTs, indicating that over half of its GT genes are candidates in the investigation of cell wall biosynthesis. Plant cell wall biosynthetic GTs use an activated form of monosaccharides, called nucleotide sugars, as donor. Monosaccharides are activated by the addition of a nucleotide, often uridine diphosphate (UDP) or guanine diphosphate (GDP). However, biochemical characterisation of cell wall GT function is made challenging by the high donor and acceptor substrate specificities ( Mariette et al., 2023 ). Furthermore, functional characterization of GTs is hampered by the genetic complexity of Arabidopsis , where multigenic families render the investigation of gene function by observing the phenotype of a simple gene knock-out difficult. Additionally, the significant capacity for compensatory wall responses from existing GTs can confuse interpretation, creating a need for a genetically simpler model organism such as the liverwort Marchantia polymorpha ( Marchantia ) to explore cell wall biosynthesis. View this table: View inline View popup Table 1. Characterised cell wall-associated GT activity across 18 families. Liverworts are a clade within Bryophytes, a monophyletic division which diverged from the land plant common ancestor around 470 million years ago ( Strother & Taylor, 2018 ). Marchantia in particular has gained traction as a popular model organism for the availability and applicability of many genetic tools, the ease of growing it in laboratories and importantly, its low genetic redundancy ( Bowman, 2022 ; Bowman et al., 2017 ; Ishizaki et al., 2016 ). For example, Marchantia has 130 protein coding genes in the 18 families where cell wall biosynthetic GTs have been identified; less than half compared to Arabidopsis. Despite having fewer genes, most cell wall polysaccharides found in land plants are conserved in Marchantia ( Happ & Classen, 2019 ; Henry et al., 2020 ; Kolkas et al., 2023 ; Pfeifer et al., 2022 ), as well as the genetics behind cell wall biosynthesis, as exemplified by the functional conservation of the RG-I rhamnosyltransferase RRT1 in Marchantia ( Wachananawat et al., 2020 ). Thus, the seemingly conserved nature of Marchantia cell wall polysaccharides and Marchantia’s low genetic redundancy makes it an attractive model organism to study the complex processes of plant cell wall polysaccharide biosynthesis. Additionally, investigating the cell wall structure in Marchantia sheds light on the lineage-specific innovations or adaptations. For example, the xyloglucans in Marchantia and mosses uniquely include the galacturonic acid containing sidechains P and Q ( Peña et al., 2008 ), and the accumulation of secondary metabolites in the cell wall, possibly to subdue environmental stresses related to terrestrialisation ( Jibran et al., 2024 ). In this study, we aim to gain a better understanding and overview of the cell wall biosynthetic precursors, GTs and polysaccharides across different Marchantia tissue types and establish Marchantia as a model organism for cell wall biosynthesis by creating a cDNA library of GTs which can be used for recombination into a variety of expression vectors to expedite functional characterisation. Furthermore, we have undertaken a comprehensive phylogenetic analysis to infer the degree of conservation and diversification of cell wall biosynthetic proteins in Marchantia . Methods Plant Material Sporangia from an Australian population of Marchantia polymorpha were collected from a southeastern Melbourne field location (37°57’48.36”S, 145° 6’20.41”E), sterilised, suspended in water and plated on petri dishes as previously described (Flores-Sandoval et al., 2015). Plants were grown in a control temperature room for the stipulated period under long day conditions following Lee et al., (2014). Glycosidic linkage Analyses Cell wall preparations (as alcohol-insoluble residue, AIR) and methylation analyses of neutral carbohydrates followed the methods described by Pettolino et al. (2012) . Briefly, dried Marchantia tissue samples were extracted consecutively with 70% (v/v) ethanol (3x), chloroform:methanol (1:1), 100% methanol and the tissue either stored in 100% ethanol or washed with 100% acetone before air drying to generate alcohol-insoluble residue (AIR). AIR samples were de-starched before performing linkage analyses. AIR preparations were carboxyl reduced to include uronic acid and methyl-esterified uronic acid residues. Monosaccharide linkage analysis was performed by multiple methylation (2x), hydrolysis, reduction and acetylation to generate partially methylated alditol acetates that were separated and identified by GC-MS. Polysaccharide levels were estimated according to Pettolino et al. (2012) by addition of appropriate deduced monosaccharide linkages for each polysaccharide based on knowledge from the wider literature, largely based on angiosperms and gymnosperms. Profiling of Nucleotide Sugars Nucleotide sugar measurements were performed as described in ( Rautengarten et al., 2019 ). In brief, 50 mg of Marchantia thallic tissue were ground in liquid nitrogen, resuspended in ice-cold methanol/chloroform (7:3), and incubated at −20 °C for 2 hours. After adding 400 µL of ice-cold water, the samples were centrifuged at 20,000 × g at 4 °C, and the upper phase was collected into 15 mL tubes on ice. The water addition and centrifugation were repeated twice, following that all supernatants were combined. Then the samples were frozen in liquid nitrogen and freeze-dried overnight. Purification was done via solid phase extraction, followed by detection and quantification of nucleotide sugars on a SCIEX 4000 QTRAP system with a TurboIonSpray source and Agilent 1100 Series Capillary LC System. Nucleotide sugar standards included UDP–α-D-xylose, UDP–β-L-arabinopyranose, UDP–α-D-galacturonic acid, UDP–α-D-glucuronic acid, UDP–α-D-glucose, UDP–α-D-galactose, UDP–N-acetyl-α-D-glucosamine, UDP–N-acetyl-α-D-galactosamine, GDP–α-D-mannose, GDP–β-L-fucose, GDP-Glc (Sigma-Aldrich), and UDP–β-L-arabinofuranose (Peptides International) ( Rautengarten et al., 2014 ). Data were analysed using Analyst 1.5.1 and quantified with MultiQuant 2.1 through the linear regression of the peak areas ( Rautengarten et al., 2016 ). Immunolabeling of Cell Wall Epitopes and Fluorescence Microscopy Fresh tissue samples were dissected and fixed and subjected to dehydration through an ethanol series. Sample were then post-processed through an ethanol-HistoClear (National Diagnostics) series in 1 hr intervals (25%, 50%, 75%, 3x 100% HistoClear) and a HistoClear-Paraplast Plus (ProSci Tech) series in half day intervals (50%, 6x 100% Paraplast Plus) with the final embedding in 100% Paraplast Plus. 6-8μm thick sections were cut using a Leica 2040 microtome and then mounted on slides. Wax was subsequently removed from the tissues with xylene washes and samples rehydrated by immersing the slides through an ethanol series with three final washes in deionized water. Immunolabelling using monoclonal antibodies LM6 and LM13, which labels short, branched arabinan and long, unbranched arabinan respectively (Verhertbruggen et al., 2009), LM15 which labels XXXG epitopes of xyloglucan ( Marcus et al., 2008 , Pedersen et al., 2012 ) (PlantProbes), BS-400-3 which labels 1-4-β-D-mannan ( Pettolino et al., 2001 ) (Biosupplies Australia) and carbohydrate binding module 3a (CBM3a) ( Hernandez-Gomez et al., 2015 ) (PlantProbes) was carried out as described previously (Lampugnani et al., 2013) except that the samples were counterstained with calcofluor and Pontamine Fast Scarlet 4B (S4B), which binds a range of β-glycans ( Anderson et al., 2010 ). Fluorescence imaging was carried out following Kesten et al. (2019) with a Nikon Eclipse Ti-E inverted Microscope fitted with a CSU-W1 Yokogawa spinning disc head and an Evolve EM-CCD camera (Photometrics Technology). Images were collected using the average of eight optical slices. Glycosyltransferase Identification and Phylogenetic Analysis To identify GTs for the phylogenetic analysis, protein sequences of M. polymorpha (MarpolBase, http://marchantia.info , TAK-1 v.7), A. thaliana (TAIR, https://www.arabidopsis.org/ ), Brachypodium distachyon (Phytozome, v3.2, The International Brachypodium Initiative, 2010), Selaginella moellendorffi (JGI PhycoCosm, v.1, Banks et al., 2011 ), Physcomitrella patens (Phytozome, v3.3, Lang et al., 2018 ) Chara braunii (NCBI, GenBank assembly: GCA_003427395.1, Nishiyama et al., 2018 ), Closterium (NCBI, Genbank assembly: GCA_949281275.1, Kawaguchi et al., 2023 ), Mesotaenium endlicherianum (JGI, SAG 12.97, Cheng et al., 2019 ) and Chlamydomonas reinhardtii (JGI, CC-4532 v6.1, Craig et al., 2023 ) were acquired from the respective sources. Orthogroups within the nine species were identified using Orthofinder v.2.5.5 ( Emms & Kelly, 2019 ), using DIAMOND as the sequence search method. Orthogroups with GT sequences were identified using Arabidopsis and Marchantia GT sequences curated in CAZy. The sequences from these orthogroups were used to construct the phylogenetic trees for the respective GT families. To construct the phylogenetic trees, the GT sequences were aligned with clustal omega (1.2.4; Sievers and Higgins, 2018) and the alignments were trimmed with trimal (1.4.1; Capella-Gutiérrez et al., 2009) using the -automated1 option. These trimmed alignments were used to build the phylogenetic trees with RaXML (8.2.12; Stamatakis, 2014) with 1000 Bootstrap using the following command: raxmlHPC -T 4 -f a -x 12345 -# 1000 -p 12345 -m PROTGAMMAAUTO -s output_trimalignment.fa -n Boot. The phylogenetic trees were visualized with iTOL (Letunic and Bork, 2021) and the figures were manually annotated. Cloning Procedures RNA was isolated from Marchantia and first-strand cDNA synthesis was carried out as described previously ( Lampugnani et al., 2016 ). Coding sequences (CDS) of target genes were retrieved from the MarpolBase ( Bowman et al., 2017 ) and gene specific primers designed for cloning into pDONR221 or alternatively pENTR d-TOPO using the Gateway Technology. Sequences of primers used in this study are shown in Table S1. Gene products were amplified using KOD Hot Start (Merck) or Q5 Hot-Start (NEB) and subsequently purified using a QIAquick PCR purification kit (Qiagen) prior to transformation. Cloned products were verified using Sanger sequencing with m13F and m13R primers. Entry clones with Mp3g24900 and Mp5g17760 coding sequences were subcloned into pEARLEYGATE101 by LR Clonase II reaction (Thermo Fisher Scientific). Nicotiana benthamiana infiltration and co-localisation Transient transformation in N. benthamiana was carried out as described in Lampugnani et al., (2016) . The Golgi and Endoplasmic Reticulum (ER) organellar markers used for co-localisation are CD3-967 and CD3-953 from Nelson et al., (2007) respectively. Results and Discussion The identification of plant cell wall biosynthetic enzymes is often hampered by high genetic redundancy in the eudicot model organism, A. thaliana . Therefore, the use of Marchantia as a genetically simpler model system could expedite the elucidation of these enzymes. To do so, we wanted to investigate the substrate availability for cell wall biosynthesis, perform an in-depth investigation of the distribution of cell wall epitopes across different tissue types in Marchantia and conduct a phylogenetic analysis of plant cell wall GT candidates. Marchantia thallus has most sugar precursors required for the biosynthesis of a typical land plant wall To examine whether Marchantia contains the corresponding substrates for the typical land plant cell wall polysaccharide biosynthesis, we conducted a nucleotide sugar analysis on mature thallus tissue. This revealed that Marchantia contains high levels of UDP-Glucose (UDP-Glc), with 50 pmol·mg -1 of fresh weight representing about 66% of the total nucleotide sugar content ( Table 2 ). As a comparison, the UDP-Glc pool is typically about 53% in Arabidopsis ( Rautengarten et al., 2014 ). Interestingly, UDP-Galacturonic acid (UDP-GalA) contents were relatively low at about 1.5 pmol·mg −1 of fresh weight representing about 1.9% of the total nucleotide sugar pool. This is about half of the amount of UDP-GalA content detected in Arabidopsis tissues ( Rautengarten et al., 2014 ). Further, there are comparatively low levels of UDP-Rhamnose (UDP-Rha) (1.7 pmol·mg −1 of fresh weight) in the Marchantia thallus when compared to Arabidopsis . As we confirm that the Marchantia thallus contains the sugar precursors required for the biosynthesis of a conventional land plant cell wall, albeit in differing proportions, we next delved into the characterisation its cell wall polysaccharide composition. View this table: View inline View popup Download powerpoint Table 2. Nucleotide sugar analysis of mature Marchantia thallus. Measurements are an average of eight technical replicates. Standard deviation values are given in brackets. The cell wall composition of Marchantia shows high pectin content in young tissues We aimed to establish the glycosyl composition of the Marchantia cell wall using linkage analysis by methylation in eight different tissue types; to investigate whether there are tissue specific differences that could highlight the importance of some polysaccharides in different biological contexts. We assayed dormant gemmae, eight-day-old gemmaling, mature thallus, antheridiophore heads, archegoniophore stalks, fertilised archegoniophore heads, sporophytes, and seven-day-old sporelings (Table S2). Linkage analysis allows the identification of individual glycosyl linkages of the wall, which can be used to predict the polysaccharide composition of the wall. However, while cell wall polysaccharides can be distinguished by their unique glycosyl linkages, it is challenging to unequivocally assign linkages to particular polysaccharides, especially in species which may have uncharacterised polymers. With this in mind, we assigned linkages largely based on cell wall polymers in other land plants ( Table 3 ). A summary of the calculated monosaccharide content of each of the tissue samples is presented in Figure S1. View this table: View inline View popup Download powerpoint Table 3. Polysaccharide deductions from the methylation analysis data according to Pettolino et al. (2012) . Abundance is colour-coded from white (lowest) to green (highest). tr = trace (<0.1%), n.d. = not detected The dominant linkage was 1,4-Glc, which made up between ∼39 to ∼57 mol% of the wall in all our samples (Table S2). This is more than what was found in Arabidopsis (33.8 mol%) ( Pettolino et al., 2012 ), but similar to what was detected in the moss P. patens (48.6 mol%) ( Moller et al., 2007 ). The 1,4-Glc linkages constitute cellulose, the backbone structure of xyloglucans and some heteromannans, such as galactoglucomannans. The β-(1,4)-Glc-linked backbone of xyloglucan is, at a minimum, substituted with α-1,6-Xyl side chains. As such, the assignment of 1,4-Glc to xyloglucan should be proportional to the number of 1,4,6-Glc linkages. Notably, the 1,4,6-Glc linkage was about 4-6 mol% in all tissues (Table S2). Mannans (β-(1,4)-linked mannan) are distinguished from (galacto)glucomannans by having only β-(1,4)-Man linkages incorporated into their backbone ( Voiniciuc et al., 2019 ; Voiniciuc, 2022 ). In the polysaccharide deduction method of Pettolino et al (2012) , the assignment of the 1,4-Glc linkage to (galacto)glucomannans hinges on the amount of 1,4-Man. In samples with rapid rates of cell division, such as the gemmae and sporelings, we detected 1,4-Man at relatively low levels, i.e. ∼1-2 mol%, compared with ∼40% mol% 1,4-Glc. In older plant samples, such as the mature thallus, antheridiophore and archegoniophore, the proportion of 1,4-Man was significantly higher, reaching up to 9 mol% in the archegoniophore stalk (Table S2). Hence, the major portion of the 1,4-Glc linkage is likely associated with cellulose and xyloglucan in younger tissues with an increasing level associated with (galacto)glucomannas in mature tissues. Assuming that the remaining 1,4-Glc linkages are assigned to cellulose, this would suggest that most Marchantia tissues have cell walls composed of 35 to 45 mol% cellulose ( Table 3 ). Another linkage that typically represents a defined polysaccharide is 1,4-Xyl. This linkage is associated with heteroxylans and its derivatives, such as (glucurono)arabinoxylan (GAX). We found that 1,4-Xyl linkages were most abundant in the sporophyte, at around 8.3 mol%, with lesser amounts in other tissue types (Table S2). Glycosyl linkages associated with arabinogalactans (AG), such as 1,4-Gal, 1,3-Gal and 1,6-Gal are present in higher levels in younger tissue types ( Table 3 ). Type I AG refers to polysaccharides with a β-1,4-Gal backbone ( Clarke et al., 1979 ; Hinz et al., 2005 ). Type I AGs are found as sidechains of RG-I and ranges in Marchantia between 0.9-10.1 mol%, with the highest levels being associated with younger tissue types. Type II AGs possess a β-1,3-Gal backbone branched with β-1,6-Gal residues, and are found on AGPs ( Clarke et al., 1979 ; Showalter, 2001 ; Seifert & Roberts, 2007 ; Tan et al., 2012 ). Like type I AGs, type II AGs range between 1.1-12.2 mol%, with the higher range associated with younger tissue types ( Table 3 ). However, the 1,4-GalA linkage, indicative of the pectic polymer homogalacturonan and RG-I, was found in low levels across all tissue types (Table S2, Table 3 ). The range of HG is estimated to be 0.4-1.9 mol%. Similarly, RG-I backbone related linkages, 1,2,4-linked Rha and equal amounts of 1,4-GalA only constituted between 0.6-4.1 mol% of the cell wall polysaccharides across the different tissue types; with highest levels associated with dormant gemmaling and cultured sporelings. The sporophyte is particularly interesting because we detected unusually high levels (∼12%) of the linkage 1,5-Ara (arabinose) (Table S2). Glycosyl linkage analysis of 7-day old sporelings allow us to draw interesting comparisons between Marchantia and another model bryophyte organism P. patens and its spore-derived tissue ( Moller et al., 2007 ). Linkage analysis of the 6-8 day old protonemal tissue of P. patens showed high levels of total GalA (∼14 mol%) compared to our results of 7-day old Marchantia sporelings (4.2 mol%), but relative Ara and Xyl levels are higher in Marchantia sporelings (7.8 mol%, 7.2 mol% respectively) than in P. patens (mol 2.5% and 4.1 mol% respectively) ( Moller et al., 2007 ). Notably, relative Gal (galactose) content is higher in sporelings (23.1 mol%) than P. patens protonema (11 mol%) ( Moller et al. 2007 ). The high abundance of Gal is also noticeable in dormant gemmae and 8-day old gemmalings (22.4 mol% and 19.4 mol% respectively), which indicate an important role of Gal-containing cell wall polysaccharides in Marchantia . Despite their shared status as early-diverging lineages of land plants, the difference in the cell wall monosaccharide composition between Marchantia and P. patens exemplifies the prevalence of taxonomic diversity in cell wall architecture. As such, while the suite of fundamental building blocks of plant cell walls is comparable across land plants, there are indeed notable differences in their relative composition. For example, dicots and non-commelinoid monocots have a cellulose and xyloglucan-dominant framework, embedded in relatively pectin-rich cell walls, also known as ‘Type I’ walls ( Carpita & Gibeaut, 1993 , Doblin et al., 2023 ). In contrast, commelinoid monocots, including the members of the Poaceae, have lower pectin content, but higher levels of the hemicellulose GAX, and are called ‘Type II’ cell walls ( Carpita and Gibeaut 1993 , Scheller and Ulvskov 2010 , Doblin et al., 2023 ). Moreover, grasses have high amounts of MLGs ( Doblin et al., 2023 ; Vega-Sánchez et al., 2013 ), basal land plants such as eusporangiates are rich in mannan ( Silva et al., 2011 , Harholt et al. 2012), and Gymnosperm cell walls are characteristic for their abundance of the hemicellulose galactoglucomannans (Voiniciuc et al. 2022). The Marchantia cell wall composition appears distinct from other land plant lineages in that its dominant hemicelluloses are xyloglucans and heteromannans and that it has lower levels of pectic polysaccharides and glycoproteins in thallus tissue, which is also consistent with the recent study by Jibran et al., (2024) . In addition, the glycosyl linkage analysis across the different life stages revealed that pectin and arabinogalactan polysaccharide content are increased in younger tissue types, and highlights the abundance of Gal-containing cell wall polysaccharides Taken together, our data indicate that Marchantia contains polysaccharide linkages representing all cell wall polymers in found in land plant walls, further supporting Marchantia as a suitable cell wall model organism for land plants. An interesting aspect of the M. polymorpha cell wall is that it lacks ‘true’ lignin in secondary cell walls ( Kremer et al., 2004 ; Pfeifer et al., 2022 ). Briefly, M. polymorpha possesses the monolignol precursors of lignin subunits p -hydroxyphenyl, guaiacyl and syringyl alcohol, and its cell wall contains high level of polyphenols ( Espiñeira et al., 2011 ). A recent study revealed that a type of flavonoid called auronidin is tightly bound to the cell wall, perhaps contributing to cell wall integrity ( Jibran et al., 2024 ). Based on the accumulation of auronidin and possibly other polyphenolic compounds, the authors speculated its role to be similar to that of lignin in tracheophytes, such as anti-microbial effects ( Carella et al., 2019 ; Humphreys et al., 2010 ), reinforcement of the primary cell wall and protection from excess light ( Jibran et al., 2024 ). In this study, only sugar-containing cell wall polymers have been considered. However, it raises the question of whether sugar-containing polymers may have to ensure certain functionalities in M. polymorpha, that in other land plants are mediated by lignin-strengthened cells. Immunolocalisation of cell wall epitopes reveals potential tissue specific functions of select cell wall polysaccharides Our polysaccharide linkage analyses gave us a comprehensive overview of the types and quantities of polysaccharides in different Marchantia tissues, however, the overview lacks information on the spatial distribution of the polysaccharides within. To further corroborate the linkage analyses and to assess where certain polysaccharides are deposited, we undertook immunofluorescence labelling experiments with antibodies that bind to specific cell wall polysaccharide epitopes. For better visualisation, sections were counter stained for better visualisation with S4B specific to cellulose ( Hoch et al., 2005 ) and calcofluor white which binds more promiscuously to several cell wall epitopes ( Anderson et al., 2010 ). To ensure that patterns of polysaccharide deposition could be compared between sample types, at least on a relative level, we adjusted the imaging settings to ensure that there were no saturated pixels in the images and maintained constant settings between sample types, which may lead to false negatives for epitopes with low abundance. As a consequence, the lack of signal in a specific tissue does not necessarily mean that the epitope is absent, as the settings may simply have been attenuated below detectable levels or some epitopes might be blocked from detection by other polymers. When the thallus was probed with either CBM3a, LM15 or BS-400-3 (β-1,4-mannan specific antibody), the corresponding signal was detected throughout the entire cross-section of the thallus, including the upper epidermis, lower epidermis, some parenchymous tissue and rhizoids, while the LM6 and LM13 arabinan antibodies were largely undetected ( Figure 1 ). We used two antibodies binding to arabinans, the LM6 antibody recognising a linear pentasaccharide in (1,5)-α-L-arabinans and the LM13 antibody recognising longer stretches of 1,5-linked arabinosyl residues that are likely to be more abundant in unbranched arabinans (Verhertbruggen et al., 2009). The absence of LM6 and LM13 binding in the rhizoids is intriguing, as LM6 shows binding to bundled rhizoids in the archegoniophore stalk ( Figure 2 ). The rhizoids are structurally intact to allow the binding of probes and antibodies in the thallus section, as shown by the binding of CBM3a and S4B. Whether there is compositional variation between the rhizoids associated with the thallus and rhizoids associated with the stalk requires further investigation. The bundled rhizoids in the stalk of the archegoniophore also showed binding of the CBM3a epitope ( Figure 2 ). LM15 xyloglucan epitopes were detected in the assimilatory filaments of the stalk, and the signal of the mannan antibody was detected extensively throughout the assimilatory filaments, bundled rhizoids and parenchymous tissue ( Figure 2E ). Download figure Open in new tab Figure 1. Immunolabelling of the Marchantia thallus with A) CBM3a (Cellulose), C) LM15 (Xyloglucan), E) β-1,4-Mannan and G) LM6 (Arabinan) I) LM13 (Arabinan) antibodies, overlayed with counterstains calcofluor white (cyan) and S4B (magenta) in B, D, F H and J respectively. ue = upper epidermis, le = lower epidermis, p = parenchymous tissue. Scale bar = 100 µm Download figure Open in new tab Figure 2. Immunolabelling of the Marchantia stalk with the A) CBM3a (Cellulose), C) LM15 (Xyloglucan), E) β-1,4-Mannan, G) LM6 (Arabinan) and I) LM13 (Arabinan) antibodies, overlayed with counterstains calcofluor white (cyan) and propodium iodide (magenta) in B, D, F, H and J, respectively. br = bundled rhizoids, af = assimilatory filaments, p = parenchyma. Scale bar = 100 µm CBM3a probes of fertilised archegoniophores localised to developing archegonia and the calyptra, a layer which protects the developing sporophyte ( Figure 3 ). LM15 bound to the assimilatory filaments, parts of the stalk and the spore jacket cells. The mannan antibody bound to the archegoniophore extensively, including the assimilatory filaments, spore jacket cells, the calyptra and the perigynium. The LM6 and LM13 antibodies recognizing arabinan epitopes showed binding to the spore capsule, developing spores as well as the foot of the sporophyte. The presence of LM6 epitopes in the sporophytes, which corroborates our glycosyl linkage analysis was also consistent with previous data ( Dierschke et al., 2024 ). A closer inspection of the sporophyte capsule showed binding of the CBM3a probe and the mannan antibody to the helical secondary cell wall structure of elaters ( Figure 4 ). While LM6 and LM13 also recognized arabinan epitopes in the elaters, with a distinct pattern from that of the CBM3a probe and the mannan antibody, in that it does not label the helical patterning of the secondary cell wall but rather co-localises to the tips of the elaters ( Figure 4E , G). The CBM3a showed binding to the assimilatory filaments and antheridial chambers of the antheridiophore ( Figure 5 ). The LM15 xyloglucan epitope was more restricted, whereas the mannan epitope was detected extensively in the antheridiophore receptacle. The LM6 and LM13 binding was weak across all cells, except for moderate LM13 binding on the dorsal surface of the antheridiophore overlapping with S4B staining. Download figure Open in new tab Figure 3. Immunolabelling of the Marchantia archegoniophore with the A) CBM3a (Cellulose), C) LM15 (Xyloglucan), E) β-1,4-Mannan and G) LM6 (Arabinan) antibodies, overlayed with counterstains calcofluor white (cyan) and S4B (magenta) in B, D, F and H respectively. c = spore capsule, ca = calyptra, p = perigynium, f = foot, af = assimilartory filaments, arrow = spore jacket cells. Scale bar = 1 mm Download figure Open in new tab Figure 4. Immunolabelling of the Marchantia sporophyte with the A) CBM3a (Cellulose), C) β-1,4-Mannan, E) LM6 (Arabinan) and G) LM13 (Arabinan) antibodies, overlayed with counterstains calcofluor white (cyan) and S4B (magenta) in B, D, F and H respectively. s = spore, e = elaters, arrowheads = tip of elaters. Scale bar = 20 µm Download figure Open in new tab Figure 5. Immunolabelling of the Marchantia antheridiophore with the A) CBM3a (Cellulose), C) LM15 (Xyloglucan), E) β-1,4-Mannan, G) LM6 (Arabinan) and I) LM13 (Arabinan) antibodies, overlayed with counterstains calcofluor white (cyan) and S4B (magenta) in B, D, F, H and J respectively. af = assimilatory filaments, ac = antheridial chambers. Scale bar = 1 mm The tissue-specific deposition of arabinan may indicate a specialised role. Previous studies have associated arabinan with a role in tip-growth, cell wall flexibility and desiccation tolerance ( Jones et al., 2003 ; Parre & Geitmann, 2005 ; Moore et al., 2008 ; Carroll et al., 2022 ). The rhizoids and elaters of Marchantia both exhibit tip growth ( Shimamura, 2016 ; Cao et al., 2019 ), and the detection of the LM6 antibody is consistent with that of other tip-growing cells such as P. patens protonemal cells and pollen tubes (K. J. D. Lee et al., 2005 ; Lampugnani et al., 2016 ). Both rhizoids (pegged) and elaters also undergo programmed cell death and desiccation ( Shimamura, 2016 ). Due to the hydrophilic properties and ability to function as a physical plasticiser, arabinan has been postulated to act as a buffer to withstand the high mechanical stresses experienced while undergoing desiccation, which is a strategy used by desiccation tolerance plants ( Moore et al., 2013 ). We therefore speculate that the deposition of arabinan in the rhizoids and elaters of Marchantia may be related to controlling the extensibility of the cell wall, and maintaining cell integrity during desiccation. However, it is uncertain which polysaccharides, if any, the arabinan is part of. The α-1,5-Ara linkage is associated with the pectic polysaccharide RG-I, AGPs ( Happ & Classen, 2019 ; K. J. D. Lee et al., 2005 ) or with a linear arabinan in a ‘free’ form that is not part of RG-I ( Lampugnani et al., 2016 ). Interestingly, LM6 does not bind to the α-1,5-linked arabinan sidechains of Marchantia AGPs (Happ and Classen, 2018) due to their short length (DP1-2), as LM6 recognizes three or more α-1,5-linked arabinosyl residues (Verhertruggen et al. 2009). Also, our glycosyl linkage analysis indicates a low proportion of predicted RG-I levels in the stalk (0.6 mol%) and sporophytes (1.3 mol%). Thus, more work is needed to conclude whether arabinan detected in the rhizoids exists in either a ‘free’ form, or associated with other polysaccharides. In combination with the glycosidic linkage results, the immunolabelling data provide a good overview of cell wall polymer distribution in Marchantia . Immunolocalization approaches using cell wall epitope directed antibodies or carbohydrate binding modules have previously shown that most polysaccharides like xyloglucan, homogalacturonan, RG-I, mannan, xylans and AGPs are somewhat conserved in Marchantia ( Happ & Classen, 2019 ; Henry et al., 2020 ; Kolkas et al., 2023 ; Dierschke et al., 2024 ; summarised in Table 4 ). Our immunolabelling results further corroborate that Marchantia indeed has the cell wall polysaccharide structures comparable to that of other land plants with a ubiquitous deposition of cellulose, xyloglucan and mannan, a targeted deposition of the pectic epitope arabinan in the gametangiophore pegged rhizoids, the sporophyte foot, capsule and elaters. Extensin and MLG antibodies were also used to probe the different structures of Marchantia , but no signal was detected ( Table 4 ). However, this does not preclude the presence of these epitopes in Marchantia , but perhaps indicate structural differences in the cell wall that hinder extensin detection or specifically in epitopes related to extensins. View this table: View inline View popup Table 4. Summary of the antibodies assayed in Marchantia polymorpha, 1 This study; Several tissue types; Immunofluorescence, 2 Happ and Classen (2019) ; AGP extract; Enzyme-linked immunosorbent assay (ELISA), 3 Kolkas et al., (2023) ; thallus cell wall extracts; Immunodot-blots, 4 Henry et al., (2020) ; Placental transfer cells of the sporophyte or gametophyte; Immunogold labelling, 5 Dierschke et al., (2024) ; Sporophytes; Immunofluorescence. Glycosyltransferase repertoire of Marchantia To further investigate the distribution of Marchantia GT genes within each family, we conducted an Orthofinder analysis using whole proteomes of A. thaliana (Dicot), B. distachyon (Monocot), S. moellendorffi (Lycophyte), M. polymorpha (Liverwort), and P. patens (Moss), C. braunii (Charales), Closterium (Desmidiales) M. endlicherianum (Zygnematales) and C. reinhardtii (Chlorophyta) (Table S3). We compiled sequences for each of the 18-cell wall-related families using the Arabidopsis and Marchantia GT list on CAZy as reference (Table S3, Figure 6 ). It should be noted that the Marchantia CAZy GT list of the 18-cell wall-related families had two gene entries duplicated (Mp1g18380 and Mp3g22290), and one gene deleted in the latest version of the Marchantia genome (TAK-1 ver.7.1). In the Arabidopsis CAZy GT list of the 18-cell wall-related families, one gene entry was triplicated (At1g53040), three gene entries were duplicated (At2g02910, At4g20170 and At4g38500. Using the sequences, we constructed a phylogenetic tree for each cell wall-related GT family, which uncovered interesting insights into the putative cell wall biosynthetic machinery in Marchantia . It should be noted that splice variants were excluded from the count only where possible ( A. thaliana, B. distachyon, P. patens and M. polymorpha ), which may have led to an overestimation of the GT numbers in the other organisms. Furthermore, reciprocal blastp analysis (a two-way blastp showing reciprocal top hits for a Marchantia gene against representative chlorophyte, streptophyte, bryophyte and tracheophyte proteomes) was conducted to infer the ancestry of GT genes within the families of interest. Download figure Open in new tab Figure 6. Number of genes in cell-wall related GT families in the species according to Orthofinder. Colour of the boxes correspond to the bar on the right-hand side. Our Orthofinder analysis identified three more orthologs in GT family 37 of Marchantia which was not listed in CAZy (Mp6g02880, Mp6g02910 and Mp6g03040). For Arabidopsis , Orthofinder identified 17 additional orthologs across families GT8 (At1g03520 and At5g30500), GT14 (At1g53100, At1g71070, At2g37585, At3g03690, At3g15350, At3g24040, At4g03340, At4g27480, At5g15050 and At5g39990), GT37 (At2g15360), GT77 (At1g14600, At2g02061 and At2g42660) and GT92 (At3g60990). Thus, when we compare the GTs in the 18-cell wall-related families, Marchantia has 130, and Arabidopsis has 288 GTs. Marchantia has fewer members than Arabidopsis in most families including major families like GT2, GT8 and GT47. In GT families 29, 34, 37 and 92, Marchantia has more members than Arabidopsis , which may indicate novel catalytic activity not present in other land plant clades rather than gene duplication with conservation of function, given the overall low genetic redundancy in Marchantia . Cellulose Marchantia only has two orthologs in the CELLULOSE SYNTHASE (CESA) subclade from GT family 2 (Figure S2). In Arabidopsis , CESAs operate in 18-subunit multimeric complexes (Cellulose Synthase Complex; CSC) ( Nixon et al., 2016 ; Purushotham et al., 2020 ). The multimeric complex consists of equimolar amounts of three different CESA homologs, which are different between primary and secondary cell wall CSCs ( Pedersen et al., 2023 ). In P. patens , there are seven CESA orthologs distributed in two subclades and both homo-oligomerisation or hetero-oligomerisation can take place ( Li et al., 2022 ). Studies of the very few orthologs in Marchantia present in the CESA clade of GT family 2 may further provide insight into the basal form of the CSC in the land plant common ancestor (Lampugnani et al., 2019). Reciprocal blast shows that Mp7g07800 and Mp2g07690 showing streptophytic ancestry with relatively high support despite losses in some embryophyte lineages (Figure S3). Hemicelluloses Members of the CELLULOSE SYNTHASE-LIKE D ( CSLD ) clade in GT family 2 exhibit β-1,4-mannan synthase and β-1,3-glucan synthase activity ( Verhertbruggen et al., 2011 ; Yang et al., 2020 ). In our phylogenetic analysis, all Marchantia members as well as the orthologs in P. patens and S. moellendorffi in the CSLD subclade are closely related to AtCSLD5 , a β-1,4-mannan synthase, and exhibit diversification within each species (Figure S2). A single Marchantia ortholog exists in the CSLA clade, where members exhibit β-1,4-mannan synthase activity ( Dhugga et al., 2004 ; Goubet et al., 2009 ; Liepman et al., 2005 ; Voiniciuc et al., 2019 ). Reciprocal blast shows that Mp2g05640 and Mp5g24530 are respectively classified as having phragmoplastidae and post-phragmoplastidae ancestry, but have been lost in all other species except for Chara (evalue = 0) and Spirogolea (evalue = 0) (Table S4). Mp5g24550 and Mp5g24550 are of embryophyte ancestry with orthologue losses in some lineages (Figure S3). Mp2g05660 is classified as bryophyte-specific but not present in Ricciocarpos . Finally, Mp2g05670 and Mp2g02440 seem to be liverwort-specific (Figure S3). The xyloglucan β-1,4-glucan synthases have been identified in CELLULOSE SYNTHASE-LIKE C (CSLC) clade in GT family 2 ( Cocuron et al., 2007 ; S.-J. Kim et al., 2020 ). Marchantia has a single ortholog in this clade (Mp8g17530), which the reciprocal blast shows is robustly of viridiplantae ancestry (Figure S3). GT34 includes xyloglucan 1,6 xylosyltransferases (XXT1-5), where XXT1 and 2 preferentially catalyse the first two Xyl substitutions of XXGG and XXXG-type xyloglucans and XXT3-5 adds the third Xyl substitution onto XXXG-type xyloglucans ( Culbertson et al., 2016 ; Ruprecht et al., 2018 ; N. Zhang et al., 2023 ). In GT34 and GT37, Marchantia and P. patens genes show extensive diversification (Figure S4, S5 respectively). The At XXT1 and 2 form a cluster distinct from At XXT3, 4 and 5. Consistent with only the XXGG branching type detected in Marchantia ( Peña et al., 2008 ), the diversified members of Marchantia are more closely related to the clade containing At XXT1 and 2 (Figure S4). Within GT34 genes, Mp3g11650 is of viridiplantae origin with orthologues in chlorophytes and streptophytes. Mp3g03380 could be of viridiplantae origin as it is found in Chlamydomonas and Ricciocarpos but has been lost in all other lineages (Table S4, Figure S6). Mp8g11830 is of streptophytic origin although it’s been lost in tracheophytes, mosses and hornworts but has orthologues in Klebsormidium and Chara . Mp2g07920 dates back to the phragmoplastidae ancestor as it is robustly found in Chara, zygnematophytes and embryophytes. Mp2g00460 is found in Ceratopteris and Ricciocarpos suggesting embryophyte origin, and Mp5g04120, Mp3g22350, Mp3g03520, Mp3g03490, Mp2g18730 seem to have orthologues only in Ricciocarpos , suggesting liverwort or marchantophyta-origin. The GT37 family contains xyloglucan α-1,2-fucosyltransferase (FUT1) which also has galactosyltransferase activity in Arabidopsis ( Cicéron et al., 2016 ; Rocha et al., 2016 ; Urbanowicz et al., 2017 ). Interestingly, P. patens and Marchantia xyloglucans do not possess the fucosylated ‘F’ sidechain ( Peña et al. 2008 ). Thus, it could be speculated that the dual function of FUT1 evolved after the split of the moss and liverwort common ancestor. On the other hand, P. patens and Marchantia xyloglucan have GalA sidechains structurally distinct from sidechains found in other land plant clades tested, including hornworts, monilophytes and lycopodiophytes ( Peña et al. 2008 ). Thus, the diversified GTs of Marchantia family 37 pose as interesting candidates to explore whether they have also independently evolved a dual function mechanism, perhaps forming the linkages in GalA-containing xyloglucan sidechains. Consistently, Mp8g06690, Mp7g18170, Mp7g07390, Mp7g06430, Mp6g10590, Mp6g02870, Mp2g22100, Mp2g21110 and Mp2g06140 are likely liverwort-specific as reciprocal blast shows only orthologues in Ricciocarpos (Table S4). Meanwhile, Mp2g16920, Mp1g01370 and Mp2g20270 have respectively streptophytic, post-phragmoplastidae (common ancestor of zygnematophycean algae and land plants) and embryophyte ancestry (Figure S7). Mp4g10040 has an orthologue only in Anthoceros suggesting bryophyte ancestry. Mp7g07400 and Mp6g19640 have orthologues in Physcomitrium but losses in Ricciocarpos suggesting setaphyte ancestry. Finally, Mp2g12440 has clear orthologues in Selaginella , Ricciocarpos and Anthoceros with a Chlamydomonas gene showing reciprocity at a high evalue threshold (evalue = 0.000733), suggesting embryophyte ancestry but possibly viridiplantae ancestry with losses in streptophytic algae. Pectic polysaccharides GT family 29 includes At GALT29A (At1g08280), a 1,3-galactan galactosyltransferase ( Dilokpimol et al. 2014 ) and the RG-II cytidine 5’-monophospho-3-deoxy-D-manno-2-octulosonic acid (CMP-Kdo) transferase ( At RCKT1; At1g08660) (Y. Zhang et al., 2024 ) (Figure S8). Phylogenetic analysis identified two putative Marchantia orthologs of At GALT29A, but none corresponding to At RCKT1. Instead, two genes, Mp5g13750 and Mp2g00070 form a distinct clade. Ancestry analysis based on reciprocal blasts, shows that Mp1g03590 is of streptophytic origin as putative orthologues are found in Klebsormidium and Chara but not in chlorophytes (Figure S9). Mp1g19260 is likely embryophyte-specific as no putative orthologues are found in chlorophytes and streptophytic algae. Mp5g13750 and Mp2g00070 are likely liverwort-specific as blastp only shows reciprocal top hits in Ricciocarpos . GT family 92 includes Arabidopsis β-1,4-galactan synthases (GALS) that are also capable of adding terminal arabinofuranose ( Liwanag et al., 2012 ; Ebert et al., 2018 ; Laursen et al., 2018 ). A clade with highly diversified Marchantia and moss sequences is present in addition to the clade containing the Arabidopsis GALS orthologs (Figure S10). Considering Marchantia has a relatively high cell wall Gal content, its members in GT92 poses an opportunity to probe for potentially new polysaccharide structures that are not present in angiosperms like Arabidopsis . Reciprocal blast suggests that Mp3g00230 and Mp1g21980 are of viridiplantae ancestry although their putative Chlamydomonas , Klebsormidium and Chara orthologues have evalues above 1xE-10 (Table S4, Figure S11). Mp6g19260 and Mp2g14970 may be of streptophyte origin although blastp evalues against Klebsormidium orthologues are respectively 6.09E-10 and 1.20E-09. Mp8g00660 is supported as of phragmoplastidae origin with Chara orthologue blastp evalues (1.07E-07) showing moderate support for this classification. Mp3g21560 and Mp2g21390 are highly supported as having post-phragmoplastidae origin with robust evalues against Spirogolea and other embryophyte proteomes but presenting losses in Ceratopteris and Arabidopsis . Mp7g09030 is likely of bryophyte origin with orthologues in Anthoceros and Physcomitrium but losses in Ricciocarpos (Figure S11). Finally Mp1g18380 seems to be of liverwort origin. The phylogenetic analysis also allows insight into the evolution of RG-II. While Marchantia has orthologs of most characterised plant cell wall related GTs, orthologs of some RG-II biosynthetic genes have not yet been identified. Specifically, members of the GT family 29 clade containing the RG-II (Kdo) transferase (RCKT1) (Y. Zhang et al., 2024 ) (Figure S8), or the RG-II xylosyltransferases (RGXT) from GT family 77 ( Egelund et al., 2006 , 2008 ; Liu et al., 2011 ; Petersen et al., 2009 ) do not have orthologs in Marchantia (Table S3). This is somewhat puzzling as orthologs of RGXT and RCKT are present in P. patens, C. braunii, Closterum and M. endlicherianum (Table S3). Furthermore, Marchantia does not have orthologs of At CDI, the putative RG-II galactosyltransferase ( Peng et al., 2021 ). Reciprocal blastp shows that Mp7g00150, Mp3g12220, and Mp1g13560 are robustly of Viridiplantae ancestry (Table S4, Figure S12). Meanwhile, Mp6g19260 and Mp2g14970 show borderline streptophytic ancestry with top hit evalues slightly above the 1xE-10 threshold in Klebsormidium . Mp8g00660 has a Chara orthologue with mild support (evalue = 1.07E-07) suggesting phragmosplastidae origin. Mp3g21560 and Mp2g21390 have clear orthologues in Spirogolea but possible losses in Arabidopsis and other embryophyte lineages, suggesting post-phragmoplastideae ancestry. Finally, Mp7g09030 and Mp1g18380 are of bryophyte and liverwort ancestry, respectively (Table S4, Figure S12). RG-II is a heterogenous pectic polysaccharide characteristic with unique sidechains which include apiose, aceric acid, 3-deoxy-D-manno-octulosonic acid (Kdo) and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha) on a 4-linked GalA backbone (Bar-Peled et al., 2012). The timing at which RG-II emerged during land plant evolution is unclear, but it is to be a land plant specific trait (Bar-Peled et al. 2012, Mikkelsen et al. 2014). Some charophycean green algae (CGA) contain Kdo and Dha either in their cell walls or associated with the cell surface (Becker et al. 1998, Domozych et al., 1991) and RG-II xylosyltransferase (RGXT) orthologs have also been identified in CGAs (Mikkelsen et al., 2014). However, whether the Kdo and Dha identified in these species is associated with RG-II, and whether the biochemical function is conserved is still unclear (Mikkelsen et al. 2014). The presence of RG-II in liverworts was speculated by the detection of, albeit in low amounts, borate-crosslinked RG-II ( Matsunaga et al., 2004 ). Furthermore, putative orthologs of the UDP-Xylose/UDP-Apiose synthase (UAS; also called AXS) in Arabidopsis are present in bryophytes ( Smith et al., 2016 , Table S4). The overexpression of the duckweed AXS1/UAS1 in the moss P. patens led to an increase in apiose content in the metabolite fraction, but was not incorporated into the cell wall, leading the authors to speculate the lack of cell wall apiosyltransferases in bryophytes and algae (J. Smith et al., 2016 ). ( Berardini et al., 2015 )Indeed, the absence of orthologs in RG-II biosynthetic GTs in Marchantia further corroborate that RG-II may be absent, or structurally distinct to RG-II characterised in spermatophytes. Despite not having orthologs of the RG-II biosynthetic GTs identified thus far, other genes that are involved in RG-II substrate biosynthesis are present in Marchantia . For example, according to TAIR ( https://www.arabidopsis.org/ ; Berardini et al., 2015 ) the GDP-D-mannose-4,6-dehydratase MURUS 1 ( MUR1 ) ( Voxeur et al., 2017 ) has four orthologs in Marchantia and one ortholog of the CMP-Kdo Synthase ( CKS1 ) ( Kobayashi et al., 2011 ). Therefore, Marchantia may be useful for gain-of-function studies for RG-II biosynthetic GTs. The reciprocal blastp analysis of Marchantia GTs reveal that the Marchantia genome has a GT repertoire that comprises ancestral streptophyte-specific, embryophyte-specific, and liverwort-specific proteins. The highest proportion of Marchantia GT-proteins show viridiplantae (N = 25/141), streptophytic (N=27), embryophytic (N = 21) and liverwort (N = 23) ancestry (Figure S13). It appears that GT-proteins have diversified across all major evolutionary transitions and they are not associated with a specific transition, perhaps suggesting the importance of consistently repurposing GT proteins to adapt to the changing environmental conditions. Building a Marchantia cell wall related GT cDNA library Given the similarities in cell wall composition and GT inventories of Marchantia and other land plants, we argued that a comprehensive Marchantia GT compendium may be a valuable resource to explore GT functions and to accelerate plant cell wall research. To this end, we attempted to clone the coding sequences (CDS) of all annotated Marchantia GTs into Gateway compatible entry vectors pDONR221 or pENTR. The use of the Gateway technology allows efficient and high throughput transfer of DNA-fragments between plasmids ( Katzen, 2007 ). All CDS sequences were amplified from mixed organ and developmental cDNA libraries using primers that placed the gene of interest in-frame with the att region and excluded the stop codon. Out of the 130 Marchantia GTs in cell-wall related families, we successfully generated 87 Gateway compatible entry vectors (Table S1). Using such an approach is advantageous as it allows entry vectors containing specific GOIs to be recombined with a range of destination vectors including those which could generate C-terminal translational fusions ( Ishizaki et al., 2015 ). To exemplify the utility of the Marchantia cell wall GT library, YFP-fusion proteins were generated with entry clones harbouring the coding sequence for the two GTs from family 47 clade G (Mp3g24900 and Mp5g17760), which were subcloned into the pEARLEYGATE101 destination vector. The two members of GT47 clade G were chosen as they appear to lack orthologs in the angiosperm model organisms in our analysis. The constructs were transiently co-expressed with Golgi apparatus and ER organelle markers in N. benthamiana. As expected for type II GTs, Mp GT47G1 (Mp3g24900) and Mp GT47G2 (Mp5g17760.1) both exhibited punctate patterns that co-localised with the Golgi-marker ( Figure 7 ). Download figure Open in new tab Figure 7. Subcellular co-localisation of Mp3g24900 and Mp5g17760.1 in N. benthamiana epidermal cells with Golgi- and ER-specific markers. Scale bar = 20 µm In conjunction with the liverwort model system that can be transformed relatively easily and grown in the laboratory, the GT library has the potential to streamline and expedite the in vivo characterisation of cell wall related GTs. Further, it will be exciting to explore the members in GT families that are highly diversified in Marchantia compared to other land plants and GTs that are specific to Marchantia . Extending from GT studies, using Marchantia as a model organism to study plant cell wall biosynthesis and delivery could be useful considering the utility of plants also for glycoengineering of complex substrates like nucleotide sugars ( Tang et al., 2023 ). Conclusion Marchantia offers a simple genetic model system to study the complex mechanisms of cell wall biosynthesis. Here, we provide a detailed characterisation of the Marchantia cell wall across different tissue types. Marchantia has a cellulose-rich cell wall with mannan and xyloglucan also being abundant but a generally low level of pectic polysaccharides in mature stages. However, increased abundance of pectic polysaccharides is observed during early developmental stages. Phylogenetic analyses of cell wall related GTs in different species across the land plant lineage reveals conservation and diversification of genes in the Marchantia family, which may allude to novel linkages that are not found in other plants. With the development of a Marchantia cell wall GT cDNA library, we hope to further accelerate the identification and characterisation of GTs involved in cell wall assembly. Conflict of Interest Statement The authors declare that they have no conflict of interests. Supplementary Figure captions S2 – S13 Figure S2. Phylogenetic tree of GT family 2. Bootstrap values above 70 are indicated with lilac circles. Splice variants are shown in black. Labelled subclades are defined based on annotated function and OrthoFinder analysis (Table S3). Figure S3. Reciprocal BLASTp analysis of Marchantia members in GT family 2. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S4. Phylogenetic tree of GT family 34. Bootstrap values above 70 are indicated with lilac circles. Splice variants are shown in black. Labelled subclades are defined based on annotated function and OrthoFinder analysis (Table S3). Figure S5. Phylogenetic tree of GT family 37. Bootstrap values above 70 are indicated with lilac circles. Splice variants are shown in black. Labelled subclades are defined based on annotated function and OrthoFinder analysis (Table S3). Figure S6. Reciprocal BLASTp analysis of Marchantia members in GT family 34. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S7. Reciprocal BLASTp analysis of Marchantia members in GT family 37. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S8. Phylogenetic tree of GT family 29. Bootstrap values above 70 are indicated with lilac circles. Splice variants are shown in black. Labelled subclades are defined based on annotated function and OrthoFinder analysis (Table S3). Figure S9. Reciprocal BLASTp analysis of Marchantia members in GT family 29. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S10. Phylogenetic tree of GT family 92. Bootstrap values above 70 are indicated with lilac circles. Splice variants are shown in black. Labelled subclades are defined based on annotated function and OrthoFinder analysis (Table S3). Figure S11. Reciprocal BLASTp analysis of Marchantia members in GT family 92. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S12. Reciprocal BLASTp analysis of Marchantia members in GT family 77. Support values represent the ratio of (sum_match/max_match), providing a measure for support for the ancestry classification (Table S4). The numbers within the tiles represent BLASTp e-values with -log10 transformation (Table S4). Figure S13. Count data from the reciprocal BLASTp analysis of Marchantia GTs. The largest proportion of Marchantia GT proteins show ancestry in Viridiplantae (N = 25/141), Streptophyta (N = 27), Embryophyta (N = 21), and liverworts (N = 23). Acknowledgements This research was supported by The University of Melbourne’s Research Computing Services and the Petascale Campus Initiative. HSK was supported by the Australian Research Training Program (RTP) scholarship and the Ruhr University Bochum PhD Exchange Scholarship. ERL and HSK are grateful for support from the University of Melbourne Botany Foundation, Vassilios Sarafis research grant and The Australia & Pacific Science Foundation. ERL acknowledges funding from the Australian Academy of Science Thomas Davies Research. BE was supported by an Australian Research Council Discovery Project (DP180102630) and the 2020 Inaugural Botany Foundation Fellowship Award during this work. SP acknowledges a Villum Investigator (Project ID: 25915), DNRF Chair (DNRF155), Novo Nordisk Laureate (NNF19OC0056076), Novo Nordisk Emerging Investigator (NNF20OC0060564), and Novo Nordisk Data Science (NNF0068884) grants. JLB and EF-S were supported by funding from the Australian Research Council (CE200100015 to JLB). The authors wish to thank Dr Uli Felzmann, The University of Melbourne’s Research Computing Services and the Petascale Campus Initiative for assistance in accessing High Performance Computing (HPC) facilities. AB, CB and MSD acknowledge the support of La Trobe University to LISAF. AB and MSD acknowledge the support of the ARC Centre in Excellence for Plant Cell Walls (CE1101007). Funder Information Declared Australian Research Training ProgramAustralian Research Training Program, , University of Melbourne Botany FoundationUniversity of Melbourne Botany Foundation, , Vassilios Sarafis research grantVassilios Sarafis research grant, , The Australia & Pacific Science FoundationThe Australia & Pacific Science Foundation, , Australian Academy of Science Thomas Davies ResearchAustralian Academy of Science Thomas Davies Research, , Australian Research Council Discovery ProjectAustralian Research Council Discovery Project, , DP180102630 Inaugural Botany Foundation Fellowship AwardInaugural Botany Foundation Fellowship Award, , Villum InvestigatorVillum Investigator, , 25915 Novo Nordisk LaureateNovo Nordisk Laureate, , NNF0068884 Novo Nordisk Emerging InvestigatorNovo Nordisk Emerging Investigator, , NNF20OC0060564 Novo Nordisk Data ScienceNovo Nordisk Data Science, , NNF0068884 Australian Research CouncilAustralian Research Council, , CE200100015 ARC Centre in Excellence for Plant Cell WallsARC Centre in Excellence for Plant Cell Walls, , CE1101007 References 1. 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