Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock.

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

Top five unanswered questions in plant cell surface research.

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

Hydroxycinnamaldehyde-derived benzofuran components in lignins.

Lignin is an abundant polymer in plant secondary cell walls. Prototypical lignins derive from the polymerization of monolignols (hydroxycinnamyl alcohols), mainly coniferyl and sinapyl alcohol, via combinatorial radical coupling reactions and primarily via the endwise coupling of a monomer with the phenolic end of the growing polymer. Hydroxycinnamaldehyde units have long been recognized as minor components of lignins. In plants deficient in cinnamyl alcohol dehydrogenase, the last enzyme in the monolignol biosynthesis pathway that reduces hydroxycinnamaldehydes to monolignols, chain-incorporated aldehyde unit levels are elevated. The nature and relative levels of aldehyde components in lignins can be determined from their distinct and dispersed correlations in 2D 1H-13C-correlated nuclear magnetic resonance (NMR) spectra. We recently became aware of aldehyde NMR peaks, well resolved from others, that had been overlooked. NMR of isolated low-molecular-weight oligomers from biomimetic radical coupling reactions involving coniferaldehyde revealed that the correlation peaks belonged to hydroxycinnamaldehyde-derived benzofuran moieties. Coniferaldehyde 8-5-coupling initially produces the expected phenylcoumaran structures, but the derived phenolic radicals undergo preferential disproportionation rather than radical coupling to extend the growing polymer. As a result, the hydroxycinnamaldehyde-derived phenylcoumaran units are difficult to detect in lignins, but the benzofurans are now readily observed by their distinct and dispersed correlations in the aldehyde region of NMR spectra from any lignin or monolignol dehydrogenation polymer. Hydroxycinnamaldehydes that are coupled to coniferaldehyde can be distinguished from those coupled with a generic guaiacyl end-unit. These benzofuran peaks may now be annotated and reported and their structural ramifications further studied.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Microparticle-mediated CRISPR DNA delivery for genome editing in poplar.

The use of CRISPR/Cas9 is currently the method of choice for precise genome engineering in plants, including in the biomass crop poplar. The most commonly used method for delivering CRISPR/Cas9 and its components in poplar is via Agrobacterium-mediated transformation, that besides the desired gene-editing event also results in stable T-DNA integration. Here we explore the delivery of the gene-editing reagents via DNA-coated microparticle bombardment into the model tree Populus tremula x P. alba to evaluate its potential for developing transgene-free, gene-edited trees, as well as its potential for integrating donor DNA at specific target sites. Using an optimized transformation method, which favors the regeneration of plants that transiently express the genes on the delivered donor DNA, we regenerated gene-edited plants that are free of the Cas9 and the antibiotic resistance-encoding transgenes. In addition, we report the frequent integration of donor DNA fragments at the Cas9-induced double-strand break, opening opportunities toward targeted gene insertions.

QT-GWAS: A novel method for unveiling biosynthetic loci affecting qualitative metabolic traits.

Although the plant kingdom provides an enormous diversity of metabolites with potentially beneficial applications for humankind, a large fraction of these metabolites and their biosynthetic pathways remain unknown. Resolving metabolite structures and their biosynthetic pathways is key to gaining biological understanding and to allow metabolic engineering. In order to retrieve novel biosynthetic genes involved in specialized metabolism, we developed a novel untargeted method designated as qualitative trait GWAS (QT-GWAS) that subjects qualitative metabolic traits to a genome-wide association study, while the conventional metabolite GWAS (mGWAS) mainly considers the quantitative variation of metabolites. As a proof of the validity of QT-GWAS, 23 and 15 of the retrieved associations identified in Arabidopsis thaliana by QT-GWAS and mGWAS, respectively, were supported by previous research. Furthermore, seven gene-metabolite associations retrieved by QT-GWAS were confirmed in this study through reverse genetics combined with metabolomics and/or in vitro enzyme assays. As such, we established that CYTOCHROME P450 706A5 (CYP706A5) is involved in the biosynthesis of chroman derivatives, UDP-GLYCOSYLTRANSFERASE 76C3 (UGT76C3) is able to hexosylate guanine in vitro and in planta, and SULFOTRANSFERASE 202B1 (SULT202B1) catalyzes the sulfation of neolignans in vitro. Collectively, our study demonstrates that the untargeted QT-GWAS method can retrieve valid gene-metabolite associations at the level of enzyme-encoding genes, even new associations that cannot be found by the conventional mGWAS, providing a new approach for dissecting qualitative metabolic traits.

Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification.

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA-containing intron. Consequently, the suppressed ccr1-6 allele was named epiccr1-6. Long-read sequencing revealed that epiccr1-6, not ccr1-6, carries dense cytosine methylation over the full length of the T-DNA. We showed that the SAIL T-DNA in the UGT72E3 locus could trigger the trans T-DNA suppression of the GABI-Kat T-DNA in the CCR1 locus. Furthermore, we scanned the literature for other potential cases of trans T-DNA suppression in Arabidopsis and found that 22% of the publications matching our query report on double or higher-order T-DNA mutants that meet the minimal requirements for trans T-DNA suppression. These combined observations indicate that intronic T-DNA mutants need to be used with caution since methylation of intronic T-DNA might derepress gene expression and can thereby confound results.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

Reassessing the claimed cytokinin-substituting activity of dehydrodiconiferyl alcohol glucoside.

Dehydrodiconiferyl alcohol glucoside (DCG) is a phenylpropanoid-derived plant metabolite with reported cytokinin-substituting and cell-division-promoting activity. Despite its claimed activity, DCG did not trigger morphological changes in Arabidopsis seedlings nor did it alter transcriptional shifts in cell division and cytokinin-responsive genes. In reinvestigating the bioactivity of DCG in its original setting, the previously described stimulation of tobacco callus formation could not be confirmed. No evidence was found that DCG is actually taken up by plant cells, which could explain the absence of any observable activity in the performed experiments. The DCG content in plant tissue increased when feeding explants with the DCG aglycone dehydrodiconiferyl alcohol, which is readily taken up and converted to DCG by plant cells. Despite the increased DCG content, no activity for this metabolite could be demonstrated. Our results therefore demand a reevaluation of the often-quoted cytokinin-substituting and cell-division-promoting activity that has previously been attributed to this metabolite.

Accelerating wood domestication in forest trees through genome editing: Advances and prospects.

The high economic value of wood requires intensive breeding towards multipurpose biomass. However, long breeding cycles hamper the fast development of novel tree varieties that have improved biomass properties, are tolerant to biotic and abiotic stresses, and resilient to climate change. To speed up domestication, the integration of conventional breeding and new breeding techniques is needed. In this review, we discuss recent advances in genome editing and Cas-DNA-free genome engineering of forest trees, and briefly discuss how multiplex editing combined with multi-omics approaches can accelerate the genetic improvement of forest trees, with a focus on wood.

Lignin engineering in forest trees: From gene discovery to field trials.

Wood is an abundant and renewable feedstock for the production of pulp, fuels, and biobased materials. However, wood is recalcitrant toward deconstruction into cellulose and simple sugars, mainly because of the presence of lignin, an aromatic polymer that shields cell-wall polysaccharides. Hence, numerous research efforts have focused on engineering lignin amount and composition to improve wood processability. Here, we focus on results that have been obtained by engineering the lignin biosynthesis and branching pathways in forest trees to reduce cell-wall recalcitrance, including the introduction of exotic lignin monomers. In addition, we draw general conclusions from over 20 years of field trial research with trees engineered to produce less or altered lignin. We discuss possible causes and solutions for the yield penalty that is often associated with lignin engineering in trees. Finally, we discuss how conventional and new breeding strategies can be combined to develop elite clones with desired lignin properties. We conclude this review with priorities for the development of commercially relevant lignin-engineered trees.

Whole genome duplication of wild-type and CINNAMYL ALCOHOL DEHYDROGENASE1-downregulated hybrid poplar reduces biomass yield and causes a brittle apex phenotype in field-grown wild types.

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both greenhouse- and field-grown trees. Strikingly, field-grown WT tetraploids developed a brittle apex phenotype, i.e., their tip broke off just below the apex. In addition, the chromosome doubling altered the biomass composition of field-grown, but not of greenhouse-grown tetraploid poplars. More specifically, the lignin content of field-grown tetraploid poplars was increased at the expense of matrix polysaccharides. This increase in lignin deposition in biomass is likely the cause of the observed brittle apex phenotype, though no major differences in stem anatomy or in mechanical properties could be found between di- and tetraploid WT poplars grown in the field. Finally, without biomass pretreatment, the saccharification efficiency of greenhouse- and field-grown WT diploids was not different from that of tetraploids, whereas that of greenhouse-grown hpCAD tetraploids was higher than that of greenhouse-grown diploids. Upon alkaline pretreatment, the saccharification yield of diploids was similar to that of tetraploids for all genotypes and growth conditions tested. This study showed that a whole genome duplication in hybrid WT and hpCAD poplar did neither result in further improvements in biomass yield, nor in improved biomass composition and, hence, saccharification performance.

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield.

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

Overexpression of the scopoletin biosynthetic pathway enhances lignocellulosic biomass processing.

Lignin is the main factor limiting the enzymatic conversion of lignocellulosic biomass into fermentable sugars. To reduce the recalcitrance engendered by the lignin polymer, the coumarin scopoletin was incorporated into the lignin polymer through the simultaneous expression of FERULOYL-CoA 6'-HYDROXYLASE 1 (F6'H1) and COUMARIN SYNTHASE (COSY) in lignifying cells in Arabidopsis. The transgenic lines overproduced scopoletin and incorporated it into the lignin polymer, without adversely affecting plant growth. About 3.3% of the lignin units in the transgenic lines were derived from scopoletin, thereby exceeding the levels of the traditional p-hydroxyphenyl units. Saccharification efficiency of alkali-pretreated scopoletin-overproducing lines was 40% higher than for wild type.

Transcriptional and metabolic changes associated with internode development and reduced cinnamyl alcohol dehydrogenase activity in sorghum.

The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Field and saccharification performances of poplars severely downregulated in CAD1.

Lignin is one of the main factors causing lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Glasshouse-grown poplars severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE 1 (CAD1), the enzyme catalysing the last step in the monolignol-specific branch of lignin biosynthesis, have increased saccharification yields and normal growth. Here, we assess the performance of these hpCAD poplars in the field under short rotation coppice culture for two consecutive rotations of 1 yr and 3 yr. While 1-yr-old hpCAD wood had 10% less lignin, 3-yr-old hpCAD wood had wild-type lignin levels. Because of their altered cell wall composition, including elevated levels of cinnamaldehydes, both 1-yr-old and 3-yr-old hpCAD wood showed enhanced saccharification yields upon harsh alkaline pretreatments (up to +85% and +77%, respectively). In contrast with previous field trials with poplars less severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD), the hpCAD poplars displayed leaning phenotypes, early bud set, early flowering and yield penalties. Moreover, hpCAD wood had enlarged vessels, decreased wood density and reduced relative and free water contents. Our data show that the phenotypes of CAD-deficient poplars are strongly dependent on the environment and underpin the importance of field trials in translating basic research towards applications.

Flexible and digestible wood caused by viral-induced alteration of cell wall composition.

Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favorable net CO2 emissions.1,2 Its potential has been demonstrated in a recent study that generated novel structural materials from flexible moldable wood.3 Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility.4 Although ARW disease is associated with the presence of an RNA virus5 known as apple rubbery wood virus (ARWV), how the unique symptoms develop is unknown. We demonstrate that the symptoms of ARWV infections arise from reduced lignification within the secondary cell wall of xylem fibers and result in increased wood digestibility. In contrast, the mid-lamellae region and xylem ray cells are largely unaffected by the infection. Gene expression and proteomic data from symptomatic xylem clearly show the downregulation of phenylalanine ammonia lyase (PAL), the enzyme catalyzing the first committed step in the phenylpropanoid pathway leading to lignin biosynthesis. A large increase in soluble phenolics in symptomatic xylem, including the lignin precursor phenylalanine, is also consistent with PAL downregulation. ARWV infection results in the accumulation of many host-derived virus-activated small interfering RNAs (vasiRNAs). PAL-derived vasiRNAs are among the most abundant vasiRNAs in symptomatic xylem and are likely the cause of reduced PAL activity. Apparently, the mechanism used by the virus to alter lignin exhibits similarities to the RNAi strategy used to alter lignin in genetically modified trees to generate comparable improvements in wood properties.6-8.

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Downregulation of barley ferulate 5-hydroxylase dramatically alters straw lignin structure without impact on mechanical properties.

Barley is a major cereal crop for temperate climates, and a diploid genetic model for polyploid wheat. Cereal straw biomass is an attractive source of feedstock for green technologies but lignin, a key determinant of feedstock recalcitrance, complicates bio-conversion processes. However, manipulating lignin content to improve the conversion process could negatively affect agronomic traits. An alternative approach is to manipulate lignin composition which influences the physical and chemical properties of straw. This study validates the function of a barley ferulate 5-hydroxylase gene and demonstrates that its downregulation using the RNA-interference approach substantially impacts lignin composition. We identified five barley genes having putative ferulate 5-hydroxylase activity. Downregulation of HvF5H1 substantially reduced the lignin syringyl/guaiacyl (S/G) ratio in straw while the lignin content, straw mechanical properties, plant growth habit, and grain characteristics all remained unaffected. Metabolic profiling revealed significant changes in the abundance of 173 features in the HvF5H1-RNAi lines. The drastic changes in the lignin polymer of transgenic lines highlight the plasticity of barley lignification processes and the associated potential for manipulating and improving lignocellulosic biomass as a feedstock for green technologies. On the other hand, our results highlight some differences between the lignin biosynthetic pathway in barley, a temperate climate grass, and the warm climate grass, rice, and underscore potential diversity in the lignin biosynthetic pathways in grasses.

Hydroxycinnamic acid-modified xylan side chains and their cross-linking products in rice cell walls are reduced in the Xylosyl arabinosyl substitution of xylan 1 mutant.

The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked l-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acid-modified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.