Stacked mutations disrupting syringyl and p-coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms

Stacked mutations disrupting syringyl and p-coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms | 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 Stacked mutations disrupting syringyl and p -coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms View ORCID Profile Pingping Ji , View ORCID Profile Osama A. Afifi , View ORCID Profile Senri Yamamoto , View ORCID Profile Yuriko Osakabe , View ORCID Profile Keishi Osakabe , View ORCID Profile Toshiaki Umezawa , View ORCID Profile Yuki Tobimatsu doi: https://doi.org/10.1101/2025.03.23.644785 Pingping Ji 1 Research Institute for Sustainable Humanosphere, Kyoto University , Gokasho, Uji, Kyoto 611-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pingping Ji Osama A. Afifi 1 Research Institute for Sustainable Humanosphere, Kyoto University , Gokasho, Uji, Kyoto 611-0011, Japan 2 Biology Department, Brookhaven National Laboratory , Upton, NY 11973-5000, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Osama A. Afifi Senri Yamamoto 1 Research Institute for Sustainable Humanosphere, Kyoto University , Gokasho, Uji, Kyoto 611-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Senri Yamamoto Yuriko Osakabe 3 School of Life Science and Technology, Institute of Science Tokyo , Kanagawa 226-8502 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuriko Osakabe Keishi Osakabe 4 Faculty of Bioscience and Bioindustry, Tokushima University , Tokushima 770-8503 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keishi Osakabe Toshiaki Umezawa 1 Research Institute for Sustainable Humanosphere, Kyoto University , Gokasho, Uji, Kyoto 611-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Toshiaki Umezawa Yuki Tobimatsu 1 Research Institute for Sustainable Humanosphere, Kyoto University , Gokasho, Uji, Kyoto 611-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuki Tobimatsu For correspondence: ytobimatsu{at}rish.kyoto-u.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary The aromatic composition of lignin significantly impacts the usability of lignocellulosic biomass. In eudicots, transgenic and mutant lines with elevated guaiacyl (G) or syringyl (S) lignin units have been successfully generated by manipulating the expression level of CONIFERALDEHYDE 5-HYDROXYLASE (CAld5H). However, this bioengineering approach has proven less effective in grasses, implicating the potential existence of a grass-specific alternative pathway for S lignin biosynthesis. Through characterization of genome-edited rice mutants, we demonstrated that S lignin in rice can be virtually eliminated by disrupting genes encoding CAld5H along with p - COUMAROYL-COENZYME A:MONOLIGNOL TRANSFERASE (PMT), a grass-specific enzyme essential for the biosynthesis of monolignol p -coumarate conjugates. In contrast, individual mutations in either CAld5H or PMT genes resulted in incomplete elimination of S lignin. These findings provide strong evidence that rice possesses a CAld5H-independent pathway leading to the grass-specific monolignol p -coumarate conjugates. In-depth structural characterizations of G-dominated lignins from rice and Arabidopsis mutants, natural gymnosperm pine, and G-type synthetic lignin revealed pronounced effects of lineage-dependent cell wall environments on the linkage patterns and molecular weight distributions of the resulting lignin polymers. Overall, our findings highlight previously overlooked lineage-specific lignin monomer biosynthesis and polymerization patterns in grasses. Introduction Grass lignocellulose, along with wood lignocellulose, represents a sustainable resource for producing biomass-based biochemicals and biofuels through biorefining. Grass biomass crops, such as sugarcane ( Saccharum spp.), sorghum ( Sorghum bicolor ), switchgrass ( Panicum virgatum ), and Miscanthus spp., are particularly valued for their high lignocellulose productivity ( Mullet et al ., 2014 ; Tye et al ., 2016 ; Umezawa et al ., 2020 ). Additionally, agricultural residues from grass grain and sugar crops offer significant potentials in utilizing them in biorefinery ( Lal, 2005 ). Compared to woody biomass, grass lignocellulosic biomass is also more amenable to chemical delignification, facilitating isolations of polysaccharide components for further downstream product development ( Davis et al ., 2013 ; Tye et al ., 2016 ; Bhatia et al ., 2017 ; Umezawa et al ., 2020 ). However, as discussed further below, the diverse and complex nature of grass lignocellulose components, particularly lignin, presents challenges in biorefinery. This underscores the importance of understanding the biosynthesis and structural characteristics of grass lignocellulose to optimize its industrial applications through molecular breeding and bioengineering approaches ( Barros and Dixon, 2020 ; Umezawa et al ., 2020 ; Chandrakanth et al ., 2023 ; Peracchi et al ., 2024 ; Umezawa, 2024 ). Lignin is an aromatic biopolymer formed through the oxidative radical coupling, or dehydrogenative polymerization, of p -hydroxycinnamyl alcohols (monolignols) and related compounds in the cell walls ( Freudenberg and Neish, 1968 ; Sarkanen and Ludwig, 1971 ; Adler, 1977 ; Higuchi, 1985 ; Ralph et al ., 2004 , 2009 , 2019 ). Lignin substructure exhibits significant variability across plant lineages, primarily due to the diversity of lignin monomers involved in lignification ( Ralph et al ., 2019 ). Generally, ferns and gymnosperms produce lignins predominantly composed of guaiacyl (G) units derived from G-type monolignol (coniferyl alcohol). On the other hand, eudicots among angiosperms incorporate both syringyl (S) units derived from S-type monolignol (sinapyl alcohol) and G units. Both gymnosperm and eudicot lignins also contain smaller amounts of p -hydroxyphenyl (H) units from H-type monolignol ( p -coumaryl alcohol). Monocotyledonous grasses among angiosperms, however, exhibit a further unique and complex lignin composition. In addition to G, S, and H units derived from the conventional (non-acylated) monolignols, grass lignins incorporate monolignol p -coumarate conjugates, such as γ- p -coumaroylated G- and S-type monolignols (coniferyl and sinapyl p -coumarate), which give rise to G and S units, respectively, appended with grass-specific p -coumarate ( p CA) units. Furthermore, grass lignins incorporate feruloylated monolignols and arabinoxylan, and the flavonoid tricin, leading to the formation of grass-specific ferulate (FA) and tricin units in the final lignin polymer ( Fig. 1 ) ( Ralph et al ., 2019 ). Download figure Open in new tab Fig. 1. Proposed lignin biosynthetic pathways in grasses. PAL, phenylalanine ammonia-lyase; PTAL, phenylalanine/tyrosine ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H/APX, 4-coumarate 3-hydroxylase/ascorbate peroxidase; 4CL, 4-coumarate:CoA ligase; C3’H, p -coumaroyl ester 3-hydroxylase; HCT, p -hydroxycinnamoyl-CoA:quinate/shikimate transferase; CSE, caffeoyl shikimate esterase; CCoAOMT, caffeoyl-CoA O -methyltransferase; CAldOMT, 5-hydroxyconiferaldehyde O -methyltransferase; COMT, caffeate O -methyltransferase; CCR, cinnamoyl-CoA reductase; HCALDH, hydroxycinnamaldehyde dehydrogenase; CAld5H, coniferaldehyde 5-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; PMT, p -coumaroyl-CoA:monolignol transferase; FMT, feruroyl-CoA:monolignol transferase; LAC, laccase; PRX, peroxidase; H, p -hydroxyphenyl units; G, guaiacyl units; S, syringyl units; P, p -coumarate units. The CAld5H (magenta) and PMT (green) of interest in this study, and the conventional monolignol biosynthetic pathways conserved in angisperms (magenta) and possible parallel monolignol p -coumarate pathways specific in grasses (green) are highlighted. Due to the significant impact of lignin aromatic composition on lignocellulosic biomass utilization, numerous efforts have been dedicated to genetically engineering lignin aromatic composition in both eudicot and grass model plants. CONIFERALDEHYDE 5-HYDROXYLASE (CAld5H or FERULATE 5-HYDROXYLASE, F5H), a cytochrome P450 (CYP) enzyme belonging to the CYP84 family, catalyzes the 5-hydroxylation step that converts G-type monolignol precursors into S-type monolignol precursors ( Humphreys et al ., 1999 ; Osakabe et al ., 1999 ) ( Fig. 1 ). This enzyme has been shown to effectively modulate the ratio of S and G lignin units, the two major aromatic types of angiosperm lignins, in several eudicot model species. For instance, down-regulation of CAld5H genes results in considerably decreased or even absent S lignin content ( Meyer et al ., 1998 ; Marita et al ., 1999 ; Reddy et al ., 2005 ; Anderson et al ., 2015 ). Conversely, over-expression of CAld5H genes increases S lignin content and in a severe case can result in an excess of 90% S lignin ( Humphreys et al ., 1999 ; Franke et al ., 2000 ; Li et al ., 2003 ; Stewart et al ., 2009 ; Anderson et al ., 2015 ). Studies also demonstrated that heterologous expression of eudicot CAld5H genes in gymnosperms, which typically produce only G lignins in the cell walls, leads to ectopic productions of S lignin in the walls ( Wagner et al . 2015 ; Edmunds et al ., 2017 ). Overall, these data strongly support the indispensable role of CAld5H in S lignin biosynthesis in eudicots. While the conservation of CAld5H in monocot grass lignin biosynthesis has been well-confirmed ( Bewg et al ., 2016 ; Takeda et al ., 2017 , 2019a ; Wu et al ., 2019 ; Tetreault et al ., 2020 ; Shafiei et al ., 2023 ), however, the capacity to modulate the S/G lignin unit ratio in grasses by manipulating CAld5H expression appears to be less straightforward and effective compared to eudicots. In rice, a loss-of-function of OsCAld5H1 , a primary CAld5H gene in rice, resulted in a predictable enrichment of G lignin units but only a partial reduction of S lignin units in major vegetative tissues. Detailed structural analyses of altered lignins produced by the CAld5H -deficient rice mutants revealed that enrichment of G units was limited to the non-γ- p -coumaroylated units, leaving grass-specific γ- p -coumaroylated lignin units largely unaffected ( Takeda et al ., 2019a ). These observations, together with supporting data from other grass mutant and transgenic lines ( Barros et al ., 2016 ; Koshiba et al ., 2017 ; Takeda et al ., 2018 ; Miyamoto et al ., 2019 ; Miyamoto et al ., 2020 ; Afifi et al ., 2022 ; Takeda-Kimura et al ., 2025 ), led to the notion that rice and possibly other grasses may possess parallel monolignol pathways independent of CAld5H activity to produce the grass-specific monolignol p -coumarate conjugates separate from the conventional non-γ- p -coumaroylated monolignols ( Takeda et al ., 2019a ; Barros and Dixon, 2020 ; Umezawa et al ., 2020 ; Peracchi et al ., 2024 ; Umezawa, 2024 ) ( Fig. 1 ). However, due to a limited understanding of these potential parallel pathways, effective regulation of the S/G lignin unit ratio in grasses has not yet been fully addressed. In this study, we aimed to develop rice mutants enriched in G lignin units by introducing stacked mutations that block both conserved and grass-specific S-type lignin monomer biosynthesis. Based on the notion that rice possesses a CAld5H -independent pathway leading to monolignol p -coumarate conjugates, we introduced loss-of-function mutations to OsCAld5H1 using CRISPR-Cas9-mediated targeted mutagenesis in a rice mutant lacking p -COUMAROYL-COENZYME A:MONOLIGNOL ACYLTRANSFERASE (PMT), a grass-specific enzyme essential for generating monolignol p -coumarate conjugates ( Withers et al ., 2012 ; Lam et al ., 2024 ) ( Fig. 1 ). As anticipated, we successfully obtained rice mutants with lignins dominated by G units and lacking S units. The structural features of the G-dominated lignins produced by these rice mutants were characterized using a comprehensive suite of analytical techniques, including chemical methods, two-dimensional (2D) nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC). These analyses were conducted comparatively with other G-dominated lignins from a eudicot CAld5H -deficient Arabidopsis mutant and natural gymnosperm pine, as well as synthetic lignin polymer (dehydrogenation polymer, DHP) prepared by in vitro polymerization of G-type monolignol (coniferyl alcohol). Taken together, our findings provide further evidence for the presence of the grass-specific parallel monolignol pathway and insights into the influence of the lineage-specific cell wall environment on lignin polymerization. Materials and Methods Plant materials oscald5h1-1 and oscald5h1-2 (identical to OsCAld5H1 -KO-a-9 and OsCAld5H1 -KO-c-1, respectively, reported in Takeda et al ., 2019a ) and ospmt1 ospmt2 (identical to ospmt1 ospmt2-2 reported in Lam et al ., 2024 ) rice mutants were obtained through CRISPR-Cas9-mediated genome editing of rice (cv. Nipponbare) as described earlier ( Toda et al ., 2019 ; Yamamoto et al ., 2024 ). To generate oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 , two single-guide RNAs (sgRNA1 and sgRNA2) targeting the first exon of OsCAld5H1 (LOC_Os10g36848) ( Fig. 2a ) were designed using the CRISPR-P 2.0 program ( Liu et al ., 2017 ) and integrated into the multiplex CRISPR-Cas9 binary vector pMgPoef4_129-2A-GFP ( Toda et al ., 2019 ; Yamamoto et al., 2024 ) using the Golden Gate Assembly Mix (New England Biolabs, USA) and the oligonucleotides listed in Table S1 . The binary vector was then transformed into embryogenic calli from ospmt1 ospmt2 via Agrobacterium tumefaciens stain EHA101 and regenerated following the methods of Hiei et al . (1994) . The regenerated T 0 plants were genotyped and grown to maturity in potting soil in a growth chamber ( Lam et al ., 2017 ). Selected T 1 plants were further genotyped and grown to maturity in a greenhouse maintained at 27 ℃ ( Lam et al ., 2017 ) along with the wild-type rice and oscald5h1-1 , oscald5h1-2 , and ospmt1 ospmt2 mutant lines. For genotyping, genomic DNA was extracted from young leaves, and the genomic region containing the target site was amplified by PCR using primers listed in Table S1 . The amplified PCR products were subjected to direct sequencing according to the method of Takeda et al . (2019a) . Arabidopsis atcald5h1 ( fah1-2 ; accession: CS6172) mutant ( Meyer et al ., 1998 ) was obtained from the Arabidopsis Biological Resource Center at Ohio University, USA, and grown to maturity in an incubator maintained at 22 °C with a 16-hour light/8-hour dark photoperiod, along with Arabidopsis Col-0 ecotype used as a wild-type control. The origin of mature Pinus taeda wood sample used in this study is detailed in Miyagawa et al . (2020) . Download figure Open in new tab Fig. 2. Generation of oscald5h1 ospmt1 ospmt2 rice mutants. (a) Gene structure and mutation patterns in the OsCAld5H1 (LOC_Os10g36848) locus of oscald5h1 (Takeda et al., 2019) and oscald5h1 ospmt1 ospmt2 (this study) mutants generated by CRISPR-Cas9-mediated targeted mutagenesis. The protospacer adjacent motif (PAM; underlined), inserted (colored in red) and deleted (colored in blue) sequences are highlighted. sgRNA1 and sgRNA2 were used to generate oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 mutants using ospmt1 ospmt2 ( Lam et al., 2024 ) as a background in this study. sgRNA3 was used to generate oscald5h1-1 and oscald5h1-2 mutants using wild-type rice as a background previously (Takeda et al., 2019). (b and c) Morphological appearance (b) and growth parameters (c) of mature wild-type and mutant (T1 homozygous lines) rice plants grown under greenhouse conditions. Scale bar = 10 cm. Values in (c) are means ± standard deviation from biological replicates ( n = 4–5). Different letters on bars indicate significant differences (one-way ANOVA with Tukey’s test, P < 0.05). WT, wild-type line; oscald5h1-1 and oscald5h1-2 , OsCAld5H1 single-knockout lines; ospmt1 ospmt2 , OsPMT1 and OsPMT2 double-knockout lines; oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 , OsCAld5H1 , OsPMT1 and OsPMT2 triple-knockout lines. Cell wall and lignin sample preparations Dried mature rice culms, Arabidopsis stems, and P. taeda wood were pulverized using a TissueLyser II (Qiagen, Hilden, Germany) shaker mill, and then sequentially extracted with water, 80% aqueous ethanol and acetone, to prepare the cell wall residue (CWR) samples used for chemical and NMR analyses ( Mansfield et al ., 2012 ). The dioxane/water-soluble lignin (DL) samples used for NMR and GPC were prepared according to the method described previously ( Martin et al ., 2023 ; Lam et al ., 2024 ). Briefly, CWR samples (approximately 1g pooled from more than 4 and 20 plants for rice and Arabidopsis, respectively) were further ball-milled using a Pulverisette7 (Fritsch Industrialist, Idar-Oberstein, Germany) planetary ball-mill in zirconium vessels containing zirconium ball bearings (600 rpm, 24 cycles of 10 min milling and 5 min break) and then subjected to digestion with crude cellulases (CELLULYSIN ® , Merk Millipore, Darmstadt, Germany). The digested CWR samples were then extracted with dioxane-water (96:4, v/v) and purified by reprecipitation in 0.01 M hydrochloric acid aqueous solution. G-type synthetic lignin (G-DHP) was prepared by in vitro polymerization of coniferyl alcohol using the end-wise polymerization method according to the previously described method ( Tobimatsu et al ., 2013 ). Chemical analysis The Klason lignin assay ( Hatfield et al ., 1994 ), quantification of cell wall-bound FA and p CA by mild alkaline hydrolysis ( Yamamura et al ., 2011 ), neutral sugar analysis by two-step trifluoroacetic acid/sulfuric acid-catalyzed hydrolysis ( Lam et al ., 2017 ), and derivatization followed by reductive cleavage (DFRC) ( Karlen et al ., 2018 ; Takeda et al ., 2018 ) were conducted according to the methods described previously. 2D NMR analysis For the 2D NMR analysis of the whole cell walls from rice stems, CWR samples (pooled from 4 biologically independent plants) were finely ball-milled using a Pulverisette7 (Fritsch Industrialist, Idar-Oberstein, Germany) planetary ball-mill following previously described conditions ( Kim and Ralph, 2010 ). For the whole cell wall NMR analysis, aliquots of the ball-milled CWR samples (approximately 60 mg) were swelled in 600 μL dimethylsulfoxide- d 6 /pyridine- d 5 (4:1, v/v) and then subjected to the 1 H– 13 C heteronuclear single-quantum coherence (HSQC) NMR analysis ( Kim and Ralph, 2010 ; Mansfield et al ., 2012 ). For the NMR analysis of DL and G-DHP samples, the samples (approximately 20 mg) were dissolved in 500 μL dimethylsulfoxide- d 6 /pyridine- d 5 (4:1, v/v) and then subjected to the 2D HSQC NMR analysis ( Martin et al ., 2023 ; Lam et al ., 2024 ). The HSQC NMR spectra were acquired using a Bruker Avance III 800 system (800 MHz; Bruker Biospin, Billerica, MA, USA) equipped with a cryogenically cooled 5 mm TCI gradient probe. Adiabatic HSQC experiments were conducted using the standard Bruker implementation (‘hsqcetgpsp.3’) and previously described parameters ( Kim and Ralph, 2010 ; Mansfield et al ., 2012 ). Data were processed and analyzed using the TopSpin 4.3 software (Bruker Biospin) as previously described ( Afifi et al ., 2022 ; Martin et al ., 2023 ; Lam et al ., 2024 ). Peak assignment was based on comparison with NMR data in literature ( Kim and Ralph, 2010 ; Mansfield et al ., 2012 ; Lan et al ., 2015 ; 2018 ; Afifi et al ., 2022 ; Martin et al ., 2023 ; Lam et al ., 2024 ; Ralph et al ., 2024 ). The central dimethylsulfoxide- d 6 solvent peaks (δ C /δ H , 39.5/2.49 ppm) were used for calibrating the chemical shift. GPC The GPC analysis of DL and G-DHP samples was conducted following the method described previously ( Tobimatsu et al ., 2013 ). Briefly, the lignin samples were dissolved in N,N -dimethylformamide with 0.1 M lithium bromide (approximately 3 mg mL −1 ), and subjected to GPC analysis on a Shimadzu LC-20AD LC system equipped with SPD-20A UV/VIS detector using the following conditions: column: Tosoh TSK gel α-M + α-2500; eluent: N,N -dimethylformamide with 0.1 M lithium bromide; flow rate: 0.5 mL min −1 ; column oven temperature: 40 °C; sample detection: UV abdorption at 280 nm. The data acquisition and computation were performed using a Shimadzu LCsolution version 5.9 software. Molecular weight calibration was conducted using the Agilent EasyVials polystyrene standards (Agilent Technologies, Santa Clara, CA, USA). Statistical analysis Student’s t test and one-way ANOVA followed by Tukey’s multiple comparison test were performed using the GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Accession numbers The accession numbers for CAld5H and PMT genes studied in this study are listed in Table S2 . Results Generation of oscald5h1 ospmt1 ospmt2 rice mutants To generated rice mutants with stacked mutations in both CAld5H and PMT genes, we introduced additional OsCAld5H1 mutations into the ospmt1 ospmt2 line, a genome-edited mutant line carrying loss-of-function mutations in OsPMT1 and OsPMT2 , two primary genes encoding PMT in rice ( Lam et al ., 2024 ). As a result, we successfully isolated two homozygous triple-knockout mutant lines, oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 , which harbor −169 bp and −7 bp deletions in the targeted OsCAld5H1 site ( Fig. 2a ). Fully genotyped oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 mutants (T 1 progenies) were grown alongside with wild-type control (WT), as well as the previously generated CAld5H -deficient oscald5h1-1 and oscald5h1-2 ( Fig. 2a ) ( Takeda et al ., 2019a ) and PMT -deficient ospmt1 ospmt2 ( Lam et al ., 2024 ) mutant lines for phenotype and cell wall characterizations ( Fig. 2b ). As previously reported ( Takeda et al ., 2019a ; Lam et al ., 2024 ), oscald5h1 and ospmt1 ospmt2 mutant lines both exhibited significant reductions in plant height and culm length, and panicle length. The stacked oscald5h1 ospmt1 ospmt2 mutant lines exhibited a similar or slightly more pronounced growth retardation phenotype compared to the oscald5h1 and ospmt1 ospmt2 mutant lines ( Fig. 2c ; Table S3 ). oscald5h1 ospmt1 ospmt2 rice produces lignin lacking S units and dominated by G units To investigate the impact of the stacked mutations in the CAld5H and PMT genes on cell wall structure, extractive-free CWR samples were prepared from mature culm tissues of the oscald5h1 , ospmt1 ospmt2 and oscald5h1 ospmt1 ospmt2 mutant lines, as well as the WT control plants, and subjected to cell wall structural analyses using chemical and NMR methods. Lignocellulose compositional analyses based on chemical analyses To investigate changes in lignocellulose composition in the rice mutants, we first examined the contents of lignin, polysaccharides, and cell-wall-bound hydroxycinnamates ( p CA and FA), in the rice culm cell walls through a series of chemical analyses. Lignin content analysis, based on the Klason lignin assay, detected no significant changes in lignin accumulation levels in the culm cell walls of most examined mutant lines, except for one of the oscald5h1 mutant lines, oscald5h1-1 , which exhibited a slight reduction ( Table 1 ). This result suggests that disruptions of either or both the CAld5H and PMT genes have a minor effect on the total lignin accumulation levels in the cell walls. The abundance of cell wall polysaccharides was investigated by performing neutral sugar analysis according to the two-step acid-catalyzed hydrolysis method. The amount of cellulosic glucose (glucose released from trifluoracetic acid-insoluble fractions) in the culm cell walls was not statistically different among the examined mutant and WT control lines ( Table 1 ). On the other hand, the amounts of hemicellulosic sugars released by trifluoroacetic acid treatment of the culm CWR samples, including glucose, xylose, and arabinose, were all significantly reduced in the PMT -deficient mutant lines, i.e., ospmt1 ospmt2 , oscald5h1-3 ospmt1 ospmt2 , and oscald5h1-4 ospmt1 ospmt2 ( Table 1 ), suggesting that disruptions of the PMT genes may impair hemicellulose biosynthesis. The accumulation levels of cell-wall-bound p CA and FA were examined by quantifying p CA and FA released upon mild alkaline hydrolysis of the culm CWR samples. As expected, the PMT -deficient ospmt1 ospmt2 , oscald5h1-3 ospmt1 ospmt2 , and oscald5h1-4 ospmt1 ospmt2 lines all displayed drastic reductions in the cell-wall-bound p CA content to undetectable levels. As previously reported for ospmt1 ospmt2 ( Lam et al ., 2024 ), these PMT -deficient lines also displayed concurrent reductions in the cell-wall-bound FA content in the culm cell walls ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Lignocellulose composition of rice culm cell wall from wild-type, oscald5h1 , ospmt1 ospmt2 and oscald5h1 ospmt1 ospmt2 . Lignin compositional analyses based on DFRC and cell wall NMR To determine the lignin monomeric composition, we first employed DFRC ( Lu and Ralph, 1997 ; 1999 ; Karlen et al ., 2018 ). The DFRC reactions cleave the major β–O–4-ether linkages in lignin polymers while leaving the γ-ester linkages intact, thereby releasing the γ- p -coumaroylated monomeric products, G′ DFRC and S′ DFRC , from the grass-specific γ- p -coumaroylated G and S units, respectively, together with non-γ-acylated monomeric products, H DFRC , G DFRC , and S DFRC , from the conventional non-γ-acylated H, G, and S units, respectively ( Fig. S1 ). The oscald5h1 lines displayed significant reductions in the S-type monomeric products, S DFRC and S′ DFRC , leading to an increase in the proportion of the DFRC-based total G units over S units in the lignin polymer. However, as we previously reported ( Takeda et al ., 2019a ), the oscald5h1 lines still contained considerable amounts of S DFRC and S′ DFRC (especially the latter; % S DFRC + % S′ DFRC = 36–39% reduced from 51% in WT), resulting in only moderate increases in the proportion of G units (% G DFRC + % G′ DFRC = 50–54% increased from 42% in WT) ( Fig. 3a ; Table S4 ). As also expected from our previous study ( Lam et al ., 2024 ), the ospmt1 ospmt2 mutant displayed eliminations of the γ- p -coumaroylated monomeric products, G′ DFRC and S′ DFRC , to undetectable levels. Particularly due to the elimination of S′ DFRC , the ospmt1 ospmt2 mutant exhibited a significant increase of the proportion of G DFRC (% G DFRC = 62%) compared to the WT control (% G DFRC Download figure Open in new tab Fig. 3. Lignin composition analysis of CAld5H- and PMT -deficient rice mutants based on DFRC and cell wall NMR. (a) Yield and composition of non-γ-acylated ( H DFRC , G DFRC and S DFRC ) and γ- p -coumaroylated ( G′ DFRC and S′ DFRC ) lignin degradation monomers released via derivatization followed by reductive cleavage (DFRC) reaction of rice culm cell wall residues (CWR). Values are means ± standard deviation from biological replicates ( n = 3). GC-MS chromatograms and yield data of the DFRC-derived monomers are shown in Fig. S1 and Table S4 . (b) Normalized signal intensities of major aromatic unit signals in 2D HSQC NMR spectra of whole rice culm cell walls. The cell wall NMR spectra, complete intergradation data, and peak assignments are shown in Fig. S2 , Fig. S3 , and Table S5 . (c) Estimations of the abundance of major lignin aromatic units (percentages of P, S, G, and H units relative to P + S + G + H = 100%) based on cell wall NMR integration data. n.d., not detected; S, syringyl units; G, guaiacyl units; H, p -hydroxyphenyl units; P, p -coumarate units; T, tricin units; F, ferulate units; WT, wild type line; oscald5h1-1 and oscald5h1-2 , OsCAld5H1 single-knockout lines; ospmt1 ospmt2 , OsPMT1 and OsPMT2 double-knockout lines; oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 , OsCAld5H1 , OsPMT1 and OsPMT2 triple-knockout lines. + % G′ DFRC = 42%) and oscald5h1 mutant (% G DFRC + % G′ DFRC = 50–54%) lines, but this mutant still contained a significant amount of S DFRC (% S DFRC = 24%) comparable to the WT level (% S DFRC = 19%) ( Fig. 3a ; Table S4 ). Strikingly, the newly developed stacked mutants, oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 lines, displayed eliminations of S DFRC (trace) along with S′ DFRC and G′ DFRC (not detected), resulting in the elimination of S-type products and a substantial increase of G-type products (% G DFRC = 81–82%) ( Fig. 3a ; Table S4 ). We also detected significant increases of H DFRC in the PMT -deficient ospmt1 ospmt2 (% H DFRC = 15%) and oscald5h1 ospmt1 ospmt2 (% H DFRC = 17-18%) mutant lines compared to the WT control (% H DFRC = 8%) and the oscald5h1 mutants (% H DFRC = 10%) ( Fig. 3a ; Table S4 ). The changes in the lignin aromatic composition in the rice mutants were further examined by cell wall NMR method. This technique involves acquiring 2D HSQC NMR spectra of ball-milled CWR samples using the direct dissolution/swelling method in a dimethylsulfoxide- d 6 /pyridine- d 5 NMR solvent system ( Kim and Ralph, 2010 ; Mansfield et al ., 2012 ). The obtained HSQC spectra of the rice cell walls displayed signals from the major lignin aromatic units, including the monolignol-derived H, G, and S units ( H , G , and S ), as well as p CA ( P ), FA ( F ) and tricin ( T ) units, which are typical of grass lignins ( Fig. S2; Table S5 ). For a semi-quantitative examination of the relative proportions of these lignin aromatic units, we performed volume integration analysis of the well-resolved lignin and p -hydroxycinnamate aromatic signals ( ½S 2/6 , G 2 , ½H 2/6 , ½P 2/6 , F 2 , and ½T 2’/6’ ) along with the major polysaccharide anomeric signals ( A 1 , U 1 , Gl 1 , X 1 , X’ 1 , and X’’ 1 ) ( Fig. S2; Table S5 ). The reported signal intensity data are relative intensities normalized to the sum of the integrated signals, approximately reflecting the proportional amount of each component in the rice cell walls ( Fig. S3 ). The obtained volume integration data further corroborated the distinct lignin compositional changes observed upon the disruptions of the CAld5H and PMT genes ( Fig. 3b,c ). In agreement with the DFRC-based lignin compositional data ( Fig. 3a ), while S units exhibited only minor decreases in the oscald5h1 mutants ( %S relative to P + S + G + H = 100 % decreased to ∼30% from 37% in WT), they were more notably decreased in ospmt1 ospmt2 ( %S = 24%) and diminished to undetectable levels in oscald5h1 ospmt1 ospmt2 along with disappearance of p CA units ( Fig. 3c ). Concurrently, the proportion of G units increased substantially in ospmt1 ospmt2 ( %G increased to 61% from 30% and 38-39% in WT and oscald5h1 , respectively) and even more significantly in oscald5h1 ospmt1 ospmt2 ( %G = 82-83%). In agreement with DFRC data ( Fig. 3a ), our NMR analysis also detected proportional increases of H units in ospmt1 ospmt2 ( %H = 10%) and oscald5h1 ospmt1 ospmt2 ( %H = 10-11%) to the WT control ( %H = 7%) and oscald5h1 lines ( %H = ∼7%) ( Fig. 3c ). Furthermore, tricin ( T ) signals were notably increased in all examined CAld5H- and PMT -deficient mutant lines ( Fig. 3b ). Notably, while we detected reductions of cell-wall-bound FA released by mild alkaline hydrolysis in the PMT -deficient mutant lines ( Table 1 ), the present NMR analysis did not support these changes ( Fig. 3b ). Collectively, the chemical and NMR data indicate that the introduction of stacked mutations in the CAld5H and PMT genes resulted in lignin predominantly composed of G units, with a virtual absence of S units. As further discussed below, these findings strongly support the existence of a CAld5H-independent parallel S lignin pathway in rice. In-depth structural characterizations of G-dominated lignins produced by oscald5h1 ospmt1 ospmt2 rice To investigate the structural features of G-dominated lignins produced by the oscald5h1 ospmt1 ospmt2 rice mutants, dioxane/water-soluble lignin (DL) samples were prepared from the culm CWR samples of the mutants and then subjected to additional lignin structural analysis using 2D HSQC NMR and GPC. For comparison, DL samples prepared from the WT control, oscald5h1 , and ospmt1 ospmt2 lines were also subjected to these analyses. Additionally, to investigate the impact of the enrichment of G lignin in rice comparatively with that in Arabidopsis, DL samples were also prepared and analyzed for the CAld5H -deficient Arabidopsis mutant atcald5h1 ( fah1-2 ) ( Meyer et al ., 1996 ; 1998 ) and its WT control (Col-0 ecotype). Furthermore, we also included a G-dominated DL sample from a gymnosperm, pine ( P. taeda ) ( Miyagawa et al., 2020 ), and a G-type synthetic lignin (G-DHP) prepared by in vitro polymerization of coniferyl alcohol ( Tobimatsu et al ., 2013 ). The aromatic composition of the plant DL and G-DHP samples was established using their 2D HSQC NMR spectra ( Fig. 4a ; Fig. S4a; Table S6 ). Volume integration analysis of the aromatic sub-regions (δ C /δ H , 150–90/8.0–6.0 ppm) estimated the proportion of G units (the percentage of G 2 relative to ½S 2/6 + G 2 + ½H 2/6 ) to have drastically increased to ∼96% in the DL samples from the oscald5h1 ospmt1 ospmt2 rice mutants from 45% in the WT control rice, 48– 51% in the oscald5h1 mutant rice, and 82% in the ospmt1 ospmt2 mutant rice. Consistent with the previous lignin composition analyses of the cell walls ( Fig. 3 ), no S and p CA units were detected in the rice oscald5h1 ospmt1 ospmt2 mutant lignins ( Fig. 4a ; Fig. S4a ). Similarly, the proportion of G units largely increased to 99% in the atcald5h1 Arabidopsis mutant from 82% in the WT control Arabidopsis ( Fig. 4a ). The G-dominated features of pine lignin (98%), and G-DHP (100%) were also confirmed ( Fig. 4a ). Download figure Open in new tab Fig. 4. 2D HSQC NMR spectra of dioxane/water-soluble lignins extracted from rice, Arabidopsis and pine, and synthetic lignin. (a) Aromatic and (b) oxygenated aliphatic sub-regions of 2D HSQC NMR spectra are displayed. The dioxane/water-soluble lignin (DL) samples were extracted from culm or stem cell walls of wild-type (WT) and oscald5h1 ospmt1 ospmt2 mutant rice, WT (Col-0 ecotype) and atcald5h1 mutant Arabidopsis, and pine ( Pinus taeda ). The synthetic lignin (G-DHP) was obtained by in vitro polymerization of coniferyl alcohol. Contour coloration matches the substructures shown in each panel. The 2D HSQC NMR spectra of DL samples from other rice lines are shown in Fig. S4 . Peak assignments are listed in Table S6 .Volume integrals are given for the major lignin aromatic units as percentages relative to the total of syringyl ( S ), guaiacyl ( G ), p -hydroxyphenyl ( H ) aromatic units (½ S 2/6 + G 2 + ½ H 2/6 = 100%) in (a) and for the major lignin inter-monomeric linkage and end-unit types as percentages relative to the total of the inter-monomeric linkage types ( I ɑ + II ɑ + ½ III ɑ + ½ IIIʹ β + IV ɑ + V ɑ = 100%) in (b). The oxygenated aliphatic sub-regions (δ C /δ H , 90–52/6.0–2.5 ppm) of the 2D HSQC NMR spectra displayed signals attributed to the major lignin inter-unit linkages, such as β–O–4 ( I ), β–5 ( II ), β–β ( III and III’ ), 5–5/β–O–4 ( IV ), and β–1 ( V ), as well as those attributed to the cinnamyl alcohol end-units ( X1 and X1’ ) ( Fig. 4b ; Fig. S4b; Table S6 ). Due to the loss-of-function of the PMT genes, the signals from the tetrahydrofuran-type β–β ( IIIʹ ) units and γ-acylated cinnamyl alcohol end-units ( Xʹ ) originating from the incorporation of γ- p -coumaroylated monolignols ( Lu and Ralph, 2002 ; Lam et al ., 2024 ) were depleted to undetectable levels in the spectra of the DL samples from the ospmt1 ospmt2 and oscald5h1 ospmt1 ospmt2 rice mutants, whereas these signals were clearly seen in the spectra of the DL samples from the WT rice and the oscald5h1 rice mutants ( Fig. 4b ; Fig. S4b ). The volume integration data of the major lignin inter-unit linkage and end-unit signals (percentages relative to I ɑ + II ɑ + ½ III ɑ + ½ III′ β + IV ɑ + V ɑ = 100%) were used to estimate the differences in the lignin linkage patterns between the lignin samples, as further reported below. Impact of G lignin enrichment in rice and Arabidopsis Compared to their corresponding WT controls, G-dominated DL samples prepared from the rice oscald5h1 ospmt1 ospmt2 and the Arabidopsis atcald5h1 mutants both exhibited notably reduced proportions of β–O–4 linkages ( I ) and increased proportions of β–5 ( II ) and 5–5/β–O–4 ( IV ) linkages ( Fig. 5a ). Such changes in lignin linkage distributions are typical consequences of G unit enrichment over S units in lignin ( Marita et al ., 1999 ; Anderson et al ., 2015 ; Takeda et al ., 2017 , 2019a , 2019b ). Indeed, these S/G composition-dependent lignin linkage shifts were clearly observed when comparing the linkage distributions between the DL samples from oscald5h1 , ospmt1 ospmt2 , and oscald5h1 ospmt1 ospmt2 mutants ( Fig. S5 ). In contrast, the rice oscald5h1 ospmt1 ospmt2 and the Arabidopsis atcald5h1 mutant DL samples exhibited different changes in the proportions of other linkage types compared to their corresponding WT control lignins: β–β linkages ( III and III’ ) showed no major changes in rice but decreased in Arabidopsis; β–1 linkages ( V ) slightly increased in rice but decreased slightly in Arabidopsis; and cinnamyl alcohol end-units ( X1 and X1’ ) decreased in rice but increased in Arabidopsis ( Fig. 5a ). Consequently, the rice mutant DL samples has notably higher amounts of β–O–4 and 5–5/β–O–4 linkages and lower amounts of β–5, β–β and β–1 linkages as well as cinnamyl alcohol end-units compared to the Arabidopsis mutant DL sample ( Fig. 5a ). GPC analysis determined that the DL samples prepared from the rice oscald5h1 ospmt1 ospmt2 and the Arabidopsis atcald5h1 mutants both have overall similar weight-averaged molecular weight ( M w ) values compared to those of the corresponding WT control DL samples ( Fig. 6a,b ; Table S7 ), suggesting that enrichment of G units over S units has a minimal impact on the molecular weights of isolated lignins in both rice and Arabidopsis. Notably, however, the rice DL samples have considerably lower molecular weights ( M w = 8,500– 9,100 Da) compared to those of the Arabidopsis DL samples ( M w = 14,500–14,900 Da) ( Fig. 6a,b ; Table S7 ). Overall, these data reveal that, despite nearly identical high proportions of G units (>96%), the rice oscald5h1 ospmt1 ospmt2 and the Arabidopsis atcald5h1 mutant lignins exhibit notably different linkage patterns and molecular weight distributions. Download figure Open in new tab Fig. 5. NMR-based lignin linkage distribution analysis of dioxane/water-soluble lignins extracted from rice, Arabidopsis and pine, and synthetic lignin. (a and b) Comparisons of volume integration data of lignin linkage types between dioxane/water-soluble lignin (DL) samples extracted from wild-type and G-lignin-dominated mutant lines of rice ( oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 ) and Arabidopsis ( atcald5h1 ) (a) and between DL samples extracted from G-lignin-dominated rice ( oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 ) and Arabidopsis ( atcald5h1 ) mutant lines, wild-type pine ( Pinus taeda ), and synthetic G lignin (G-DHP) prepared by in vitro polymerization of coniferyl alcohol (b). The HSQC NMR spectra of the DL samples and peak assignments are shown in Fig. S4 and Table S6 . Volume integral analysis was performed for the major lignin inter-monomeric linkage and end-unit types as percentages relative to the total of the analyzed inter-monomeric linkage types ( I ɑ + II ɑ + ½ III ɑ + IV ɑ + V ɑ = 100%). (c) Expanded β–O–4 correlation sub-regions of 2D HSQC spectra of lignin samples, highlighting I β -correlations from β–O–4 linkages linked to G ( I G ), tricin ( I T ), and ferulate ( I F ) units ( Lan et al ., 2018 ). Download figure Open in new tab Fig. 6. Molecular weight distributions and 3D models of dioxane/water-soluble lignins extracted from rice, Arabidopsis and pine, and synthetic lignin. Molecular weight distribution curves (a), molecular weight distribution data (b), and representative 3D structure models (c) of dioxane/water-soluble lignin (DL) samples extracted from G-lignin-dominated rice ( oscald5h1-3 ospmt1 ospmt2 and oscald5h1-4 ospmt1 ospmt2 ) and Arabidopsis ( atcald5h1 ) mutant lines, wild-type pine ( Pinus taeda ), and synthetic G lignin (G-DHP) prepared by in vitro polymerization of coniferyl alcohol. Molecular weight distribution data were obtained by gel permeation chromatography (GPC) using polystyrene molecular weight standards and complete data are listed in Table S7 . M n , number-averaged molecular weight; M w , weight-averaged molecular weight; PDI, polydiversity index ( M w / M n ). To generate 3D model structures in (c), SMILES strings were created using LigninKMC ( Orella et al., 2019 ) based on aromatic composition and bonding patterns derived from 2D HSQC NMR and molecular weight distribution data from GPC. These SMILES strings were then used for structural modeling and optimization with LigninBuilder ( Vermaas et al., 2018 ) and VMD ( Humphrey et al., 1996 ) to obtain average lignin models. Finally, PyMOL (2.5 Schrödinger-LLC) was employed for visualization. Comparison among G-dominated lignins produced in different plant lineages and G-DHP The notable structural differences between the G-dominated rice and Arabidopsis mutant DL samples prompted us to expand the comparative analysis to include natural G-dominated pine DL and G-type synthetic lignin G-DHP. As previously established in earlier DHP studies ( Ralph et al ., 2004 ; 2009 ), G-DHP exhibited a markedly distinct linkage pattern compared to that of the other plant DL samples examined. It displayed a much lower content of β–O–4 linkages ( I ) and considerably higher contents of β–5 ( II ) and β–β ( III ) linkages, as well as cinnamyl alcohol end-units ( X1 ), compared to those observed in the rice, Arabidopsis and pine DL samples ( Fig. 5b ). These differences can be attributed to the critical difference between the lignin polymerization environments in vitro and in vivo ( Ralph et al ., 2004 ; 2009 ), as further discussed below. Meanwhile, the G-dominated DL samples from rice, Arabidopsis and pine also exhibited notable variations in their linkage patterns. Rice DL is characterized by their higher contents of β–O–4 linkages ( I ); the Arabidopsis DL is distinguished by its higher contents of β–5 ( II ), β–β ( III ) and β–1 ( V ) linkages as well as cinnamyl alcohol end-units ( X1 ); and pine DL is characterized by its higher content of 5–5/β–O–4 linkages ( IV ) compared to others ( Fig. 5b ). In addition, GPC analysis determined that the rice mutant DL samples showed notably lower molecular weights ( M w = 8,500 Da) compared to those of Arabidopsis ( M w = 14,900 Da) and pine ( M w = 12,300 Da) DL samples as well as G-DHP ( M w = 12,200) ( Fig. 6a,b ; Table S7 ). These structural differences between the G-dominated plant lignins (3D models illustrated in Fig. 6c ) may reflect variations in the cell wall environment in different plant lineages that influence the polymerization of the G-type monolignol, coniferyl alcohol, during cell wall lignification, as further discussed below. Discussion Complete elimination of S lignin by stacked mutations to CAld5H and PMT provides further evidence for parallel monolignol pathways in grasses In the present study, we demonstrated that CAld5H and PMT cooperatively act to generate S lignin in grass rice. As previously demonstrated ( Takeda et al ., 2019a ), loss-of-function mutations in OsCAld5H1 , a primary gene encoding CAld5H in rice, significantly reduce but do not eliminate S units, resulting in the persistence of substantial amounts of both non-acylated and γ- p -coumaroylated S units, especially the latter ( Fig. 3 ). This sharply contrasts with the complete absence of S lignin in the CAld5H -deficient eudicot Arabidopsis mutant atcald5h1 ( Meyer et al ., 1998 ; Marita et al ., 1999 ; Anderson et al ., 2015 ) ( Fig. 4 ). Loss-of-function mutations in OsPMT1 and OsPMT2 , two primary genes encoding PMT in rice ( Lam et al ., 2024 ), also significantly reduce but do not eliminate S lignin, leaving non-acylated S units present in the mutant lignin ( Fig. 3 ). Strikingly, the introduction of stacked mutations to both CAld5H and PMT genes resulted in the near-complete absence of S units in rice lignin ( Fig. 3 ). This finding strongly supports the notion that rice possesses a parallel CAld5H -independent pathway, in which PMT plays an essential role in generating the S-type monolignol p -coumarate conjugate (sinapyl p -coumarate) alongside the conventional CAld5H-dependent pathway toward the conventional (non-γ-acylated) S-type monolignol (sinapyl alcohol) ( Takeda et al ., 2019a ) ( Fig. 1 ). Furthermore, this study demonstrates that targeting both CAld5H and PMT genes simultaneously provides a viable strategy for precisely manipulating lignin aromatic composition in rice and potentially other grasses. The question of whether such CAld5H-independent monolignol pathway proposed in rice are widely present is other grass species is yet to be determined. This is particularly because most functional characterizations of CAld5H genes in other grasses to date have relied on incomplete downregulation of the genes using RNAi approaches. In sugarcane, RNAi-mediated downregulation of CAld5H genes resulted in a partial decrease in the S:G lignin unit ratio, from 61:39 to 48:52, with no detectable influence on cell-wall-bound p CA as determined by 2D HSQC NMR ( Bewg et al ., 2016 ). Similarly, in switchgrass ( Panicum virgatum L. ), RNAi-mediated downregulation of CAld5H genes, in combination with genes encoding 5-HYDROXYCONIFERALDEHYDE O -METHYLTRANSFERASE (CAldOMT), resulted in only partial reductions in S units in lignin compared to wild-type plants ( Wu et al ., 2018 ). In another recent study on barley ( Hordeum vulgare ), downregulation of HvF5H1 , a primary barley CAld5H gene, by RNAi led to a substantial reduction in S units in lignin, with the S/G ratio reduced by up to ∼85% from 1.62 in a control plant to 0.24. Intriguingly, this reduction in S lignin content accompanied with significant reductions in cell-wall-bound p CA, raising questions about the existence of the parallel monolignol pathway in barley ( Shafiei et al ., 2023 ). However, to conclusively determine whether the parallel monolignol pathway analogous to that proposed in rice exist in these grass species, further lignin characterization of mutants with loss-of-function in CAld5H is necessary. The findings in rice mutants lacking CAld5H resemble those in rice transgenic lines in which a single functional gene ( OsC3’H1 ) encoding p -COUMAROYL ESTER 3-HYDROXYLASE (C3′H) was downregulated ( Takeda et al ., 2018 ; Takeda-Kimura et al ., 2025 ). As C3′H catalyzes the first aromatic m -hydroxylation step in monolignol biosynthesis ( Fig. 1 ), the downregulation of C3′H genes typically leads to an overaccumulation of H units with a corresponding decrease in G and S units in lignin ( Franke et al ., 2002 ; Ralph et al ., 2006 ; Bonawitz et al ., 2014 ; Takeda et al ., 2018 ; Takeda-Kimura et al ., 2025 ). However, similar to the increase of S units in the CAld5H -deficient rice ( Takeda et al ., 2019a ; this study) ( Fig. 3 ), the increase in H units in the C3’H -deficient rice only partially occurred primarily at the expense of non-γ- p -coumaroylated G and S units, while the levels of γ- p -coumaroylated G and S units remained largely unaffected ( Takeda et al ., 2018 ; Takeda-Kimura et al ., 2025 ). This finding strongly suggests the existence of a C3′H-independent parallel pathway that synthesizes γ- p -coumaroylated monolignols separately from non-γ- p -coumaroylated monolignols, similar to the CAld5H-independent pathway explored in this study. Combined with the results of the rice mutants lacking CAld5H and PMT ( Takeda et al ., 2019a ; Lam et al ., 2024 ; this study), it is probable that, as illustrated in Fig. 1 , rice may possess the capability to perform aromatic methoxylation ( m -hydroxylation followed by O -methylation) of γ- p -coumaroylated monolignols, utilizing unidentified hydroxylase(s) [and O -methyltranferase(s)] and that function independently of CAld5H and C3’H. The possible role of the H-type γ- p -coumaroylated monolignol as a precursor for the G- and S-type γ- p -coumaroylated monolignols was also suggested based on the high catalytic activity of rice PMT (OsPMT1/OsAT4) toward the H-type monolignol ( p -coumaryl alcohol) in vitro ( Withers et al ., 2012 ). However, future studies aimed at identifying missing hydroxylase and O -methyltransferase enzymes involved in the conversions of γ- p -coumaroylated monolignols are crucial for validating this possibility. Several other studies have also implicated the possible existence of the alternative monolignol pathways towards grass-specific lignin monomers. For instance, it was reported that the function of the grass-specific bifunctional PHENYLALANINE/TYROSINE AMMONIA-LYASE (PTAL) ( Fig. 1 ) in Brachypodium distachyon may be more specifically linked to the productions of S and p CA lignin units derived from sinapyl p -coumarate ( Barros et al ., 2016 ; Barros and Dixon, 2020 ). Two different isoforms of the rice 4-COUMAROYL-COENZYME A LIGASE (Os4CL3 and Os4CL4) ( Fig. 1 ) have been reported to differentially contribute to the biosynthesis of monolignol and monolignol p -coumarate conjugates as well as tricin ( Afifi et al ., 2022 ). Moreover, OsMYB108, a rice R2R3-MYB transcription factor that negatively regulates lignin biosynthesis, is more strongly involved in the biosynthesis of the grass-specific lignin monomers, monolignol p -coumarate conjugates and tricin, compared to its involvement in the biosynthesis of the conventional non-γ- p -coumaroylated monolignols ( Miyamoto et al ., 2019 ; Miyamaoto et al ., 2020). Overall, the accumulated data collectively underscore the complexity of the metabolic network responsible for the biosynthesis of diverse lignin monomers in grasses, which substantially differs from that observed in eudicots ( Barros and Dixon, 2020 ; Chandrakanth et al ., 2023 ; Peracchi et al ., 2024 ; Umezawa et al ., 2020 , 2024). A comprehensive understanding of this network requires further study of monolignol biosynthetic enzymes and genes, particularly those potentially involved in producing monolignol derivatives independent of CAld5H and C3’H in grasses ( Fig. 1 ). Distinct structural features of G-dominated lignins from rice, Arabidopsis and pine highlight lineage-dependent lignin polymerization patterns The successful elimination of S units along with p CA units in rice lignin allowed us to examine the structural characteristics of G-dominated lignins produced in rice (a herbaceous monocot grass), along with those produced in Arabidopsis (a herbaceous eudicot) and pine (a woody gymnosperm). Previous studies on lignin polymerization, primarily utilizing in vitro systems to prepare DHPs, have identified numerous factors that can potentially influence lignin polymer formation in planta . These factors include monomer supply rate, pH, ionic strength, the capacity of polymerization enzymes (laccases and peroxidases), the influence of polysaccharide matrices, etc. in lignifying cell walls where lignin monomers undergo combinatorial radical coupling (as intensely discussed, for example, in Terashima et al ., 1995 ; Brunow et al ., 1998 ; Cathala et al ., 1998 ; Syrjänen and Brunow, 2000 ; Ralph et al ., 2004 ; Méchin et al ., 2007 ; Ralph et al ., 2009 ; Tobimatsu et al ., 2010 ; Hwang et al ., 2015 ; Li et al ., 2015 ; Tobimatsu and Schuetz, 2019 ; Tokunaga and Watanabe, 2023 ). Thus, the observed structural variations in the G-dominated plant lignins could be attributed to differences in cell wall environmental factors that modulate the polymerization of the G-type monolignol, coniferyl alcohol. Consistent with earlier DHP studies ( Sarkanen and Ludwig, 1971 ; Ralph et al ., 2004 ; 2009 ), G-DHP displayed considerably different structural features compared to the G-dominated plant lignins, with a markedly low level of β–O–4 linkages and high levels of β–5, β–β, and cinnamyl alcohol end-unit linkages ( Fig. 5 ). These distinct differences between DHP and plant lignins can be primarily attributed to the over-representation of dehydrodimerization reactions over cross-coupling reactions in typical DHP preparation conditions. While dehydrodimerization reactions of coniferyl alcohol typically yield β–O–4, β–5 and β–β dimers at comparable levels, cross-coupling reactions of coniferyl alcohol with growing lignin polymers favor the formation of β–O–4 linkages ( Syrjänen and Brunow, 2000 ; Ralph et al ., 2004 ; 2009 ). Nevertheless, despite exhibiting general structural similarities with each other when compared to G-DHP, our NMR and GPC analyses detected notable structural differences among the three G-dominated plant lignins in terms of their lignin linkage distributions ( Fig. 5 ) and molecular weight distributions ( Fig. 6 ). The G-dominated lignins isolated from the rice oscald5h1 ospmt1 ospmt2 mutants were characterized by their relatively higher contents of β–O–4 linkages and also their lower molecular weights compared to those from Arabidopsis and pine ( Fig. 5 ; Fig. 6 ). These differences may be attributed, at least in part, to the presence of grass-specific tricin and ferulate nucleation sites within grass cell walls, potentially influencing lignin polymerization initiation. Tricin, a canonical lignin monomer in grasses, has been shown to exclusively cross-couple with monolignols and γ-acylated monolignols via the β–O–4-type coupling mode (4ʹ–O–β-coupling) both in vivo and in vitro ( del Río et al ., 2012 ; 2020; Lan et al ., 2015 ; Elder et al ., 2020 ). Since tricin is unable to participate in homocoupling or direct cross-coupling with growing lignin polymers, it preferentially incorporates into the starting point of nascent lignin polymer chains (via cross-coupling with other lignin monomers), thereby serving as an initiation site for lignin polymerization ( Lan et al ., 2015 ; del Río et al ., 2020 ; Berstis et al ., 2021 ; Lam et al ., 2021 ). Similarly, ferulate, which is predominantly esterified to arabinoxylan, serves as an initiation site for lignin polymerization and preferentially cross-couples with monolignols via the β–O–4 linkage during cell wall lignification in grasses ( Bunzel et al ., 2004 ; Ralph, 2010 ). Therefore, the abundant presence of tricin and ferulate initiation sites in grass cell walls may contribute to the increased formation of β–O–4 linkages in lignin, as observed in the G-dominated rice lignin in the present study. Supporting this idea, we found that a significant portion of the β–O–4 linkages in the rice mutant lignins originated from the cross-coupling of coniferyl alcohol with tricin ( Fig. 5c ). The abundant presence of tricin and ferulate initiation sites may also contribute to limiting the growth of lignin polymer chains in grass cell walls. This occurs because the available pool of coniferyl alcohol is distributed among a larger number of growing polymer chains originating from abundant initiation sites, reducing the amount of coniferyl alcohol available for the elongation of each individual chain. The G-dominated lignin from the Arabidopsis atcald5h1 mutant exhibited “bulk polymer” features more closely resembling G-DHP, with a lower content of β–O–4 linkages and higher contents of β–5, β–β, and cinnamyl alcohol end-unit linkages compared to the other G-dominated lignins from rice and pine ( Fig. 5 ). Since such “bulk polymer” features are well-known to be associated with rapid monomer supply rates during lignification ( Sarkanen and Ludwig, 1971 ; Ralph et al ., 2004 ; 2009 ; Tokunaga and Watanabe, 2023 ), the relatively fast lignification process during stem development in Arabidopsis may promote the formation of bulk-polymer-type linkages in lignin. On the other hand, the G-dominated lignin from pine exhibited higher amounts of 5–5/β–O–4 linkages ( Fig. 5 ). Since the formation of 5–5 linkages primarily occurs through cross-coupling reactions between polymer/oligomer chains during the later stages of lignin polymerization ( Sarkanen and Ludwig, 1971 ; Brunow et al ., 1998 ; Ralph et al ., 2004 ; 2009 ; Tokunaga and Watanabe, 2023 ), this observation suggests that, in contrast to the rapid Arabidopsis lignification as proposed above, the relatively slower, long-term lignification process during pine wood formation might favor the preferential occurrence of 5–5 linkages in pine lignin. One of the apparent differences in lignin polymerization among the three plant species examined in this study is the distinct composition of their cell wall polysaccharide matrices. Specifically, the cell wall polysaccharide matrices in rice (grass), Arabidopsis (eudicot), and pine (gymnosperm) are predominantly composed of arabinoxylan, glucuronoxylan, and glucomannan, respectively ( Scheller and Ulvskov, 2010 ). As lignin polymerization in planta occurs after the deposition of polysaccharides in developing cell walls, pre-existing polysaccharide matrices may act as scaffolds for lignin polymerization, providing specific local environments wherein lignin monomers undergo radical coupling reactions. Indeed, prior research has demonstrated that DHP formation can be affected by the presence of various polysaccharides in the polymerization system ( Terashima et al ., 1995 ; Cathala and Monties, 2001 ; Nakamura et al ., 2006 ; Barakat et al ., 2007 ; Li et al ., 2015 ; Warinowski et al ., 2016 ; Aminzadeh et al ., 2017 ). However, the influence of varying polysaccharide matrices on lignin linkage patterns is still not well-defined. It would therefore be intriguing to revisit DHP preparations in the presence of different polysaccharide matrices and analyze the resultant DHP structures using advanced lignin characterization techniques. Further in-depth comparative structural characterizations of lignins with well-defined monomer composition, generated both in vivo and in vitro , will be crucial for advancing our understanding of the factors controlling lignin polymerization in planta . Such studies will ultimately enhance our ability to manipulate lignin structure for improved lignocellulose utilization. Competing interests The authors declare that they have no competing interests. Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Author contributions P.J., T.U. and Y.T. conceived research. P.J., O.A.A, S.Y., Y.O., K.O. and Y.T. performed experiments, and analyzed the data. P.J. and Y.T. wrote the manuscript with help from all the others. Supporting Information Fig. S1 DFRC-derived lignin monomers released from rice cell walls. Fig. S2 2D HSQC NMR spectra of whole rice cell walls. Fig. S3 Volume integration analysis of 2D HSQC NMR spectra of rice cell walls. Fig. S4 2D HSQC NMR spectra of dioxane/water-soluble lignins. Fig. S5 NMR-based lignin linkage distribution analysis of dioxane/water-soluble lignins. Table S1 Primers and oligonucleotides used in this study. Table S2 Accession numbers of CAld5H and PMT genes studied. Table S3 Growth characteristics of rice mutants. Table S4 Yield and composition of DFRC-derived lignin monomers. Table S5 Peak assignments in 2D HSQC NMR spectra of rice cell walls. Table S6 Peak assignments in 2D HSQC NMR spectra of dioxane/water-soluble lignins. Table S7 Molecular weight distribution data of dioxane/water-soluble lignins and synthetic lignin. Acknowledgments We thank Prof. Hironori Kaji and Ms. Ayaka Maeno (ICR, Kyoto University) for their assistance in NMR analysis. This work was supported in part by grants from the Japan Society for the Promotion of Science (grant nos. KAKENHI #JP20H03044 and #JP24K01827). 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Share Stacked mutations disrupting syringyl and p -coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms Pingping Ji , Osama A. Afifi , Senri Yamamoto , Yuriko Osakabe , Keishi Osakabe , Toshiaki Umezawa , Yuki Tobimatsu bioRxiv 2025.03.23.644785; doi: https://doi.org/10.1101/2025.03.23.644785 Share This Article: Copy Citation Tools Stacked mutations disrupting syringyl and p -coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms Pingping Ji , Osama A. Afifi , Senri Yamamoto , Yuriko Osakabe , Keishi Osakabe , Toshiaki Umezawa , Yuki Tobimatsu bioRxiv 2025.03.23.644785; doi: https://doi.org/10.1101/2025.03.23.644785 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7643) Biochemistry (17717) Bioengineering (13910) Bioinformatics (42015) Biophysics (21477) Cancer Biology (18626) Cell Biology (25536) Clinical Trials (138) Developmental Biology (13392) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22530) Immunology (17755) Microbiology (40437) Molecular Biology (17200) Neuroscience (88703) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4832) Physiology (7657) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9828) Zoology (2272)