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The applicability of the electronegatively equalization method (EEM) is investigated for the calculation of atoms-in-molecules charges in prototypical organic molecules. The EEM approach is a powerful way to obtain AIM charges without the computational cost of the ab initio approach.

Lignin is a phenolic polymer in plants that rigidifies the cell walls of water-conducting tracheary elements and support-providing fibers and stone cells. Different mechanisms have been suggested for the transport of lignin precursors to the site of lignification in the cell wall. Extracellular vesicle (EV)-enriched samples isolated from a lignin-forming cell suspension culture of Norway spruce (Picea abies L. Karst.) contained both phenolic metabolites and enzymes related to lignin biosynthesis. Metabolomic analysis revealed mono-, di-, and oligolignols in the EV isolates, as well as carbohydrates and amino acids. In addition, salicylic acid (SA) and some proteins involved in SA signaling were detected in the EV-enriched samples. A proteomic analysis detected several laccases, peroxidases, β-glucosidases, putative dirigent proteins, and cell wall-modifying enzymes, such as glycosyl hydrolases, transglucosylase/hydrolases, and expansins in EVs. Our findings suggest that EVs are involved in transporting enzymes required for lignin polymerization in Norway spruce, and radical coupling of monolignols can occur in these vesicles., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Forage maize is a versatile crop extensively utilized for animal nutrition in agriculture and holds promise as a valuable resource for the production of fermentable sugars in the biorefinery sector. Within this context, the carbohydrate fraction of the lignocellulosic biomass undergoes deconstruction during ruminal digestion and the saccharification process. However, the cell wall's natural resistance towards enzymatic degradation poses a significant challenge during both processes. This so-called biomass recalcitrance is primarily attributed to the presence of lignin and ferulates in the cell walls. Consequently, maize varieties with a reduced lignin or ferulate content or an altered lignin composition can have important beneficial effects on cell wall digestibility. Considerable efforts in genetic improvement have been dedicated towards enhancing cell wall digestibility, benefiting agriculture, the biorefinery sector and the environment. In part I of this paper, we review conventional and advanced breeding methods used in the genetic improvement of maize germplasm. In part II, we zoom in on maize mutants with altered lignin for improved digestibility and biomass processing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vanhevel, De Moor, Muylle, Vanholme and Boerjan.)

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., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hoengenaert, Van Doorsselaere, Vanholme and Boerjan.)

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., Competing Interests: Conflict of interest statement. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shafiei, Hooper, McClellan, Oakey, Stephens, Lapierre, Tsuji, Goeminne, Vanholme, Boerjan, Ralph and Halpin.)

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., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Meester, Oyarce, Vanholme, Van Acker, Tsuji, Vangeel, Van den Bosch, Van Doorsselaere, Sels, Ralph and Boerjan.)

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., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as 'good neighbors' in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of 'good neighbors' in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural characterization of 427 of the 5,420 profiled compounds, including phenylpropanoids, flavonoids, benzoxazinoids, and auxin-related compounds, among others. Only 75 of the 427 compounds were already described in maize. Analysis of the CSPP networks showed that phenylpropanoids are present in all organs, whereas other metabolic classes are rather organ-enriched. Frequently occurring CSPP mass differences often corresponded with glycosyl- and acyltransferase reactions. The interplay of glycosylations and acylations yields a wide variety of mixed glycosides, bearing substructures corresponding to the different biochemical classes. For example, in the tassel, many phenylpropanoid and flavonoid-bearing glycosides also contain auxin-derived moieties. The characterized compounds and mass differences are an important step forward in metabolic pathway discovery and systems biology research. The spectral metadata of the 5,420 compounds is publicly available (DynLib spectral database, https://bioit3.irc.ugent.be/dynlib/)., (© 2021 The Author(s).)

Lignin causes lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Engineered low-lignin plants have reduced recalcitrance but often exhibit yield penalties, offsetting their gains in fermentable sugar yield. Here, CRISPR/Cas9-generated CCR2(-/*) line 12 poplars have one knockout CCR2 allele while the other contains a 3-bp deletion, resulting in a 114I115A-to-114T conversion in the corresponding protein. Despite having 10% less lignin, CCR2(-/*) line 12 grows normally. On a plant basis, the saccharification efficiency of CCR2(-/*) line 12 is increased by 25-41%, depending on the pretreatment. Analysis of monoallelic CCR2 knockout lines shows that the reduced lignin amount in CCR2(-/*) line 12 is due to the combination of a null and the specific haploinsufficient CCR2 allele. Analysis of another CCR2(-/*) line shows that depending on the specific CCR2 amino-acid change, lignin amount and growth can be affected to different extents. Our findings open up new possibilities for stably fine-tuning residual gene function in planta.

PIRIN (PRN) genes encode cupin domain-containing proteins that function as transcriptional co-regulators in humans but that are poorly described in plants. A previous study in xylogenic cell cultures of Zinnia elegans suggested a role for a PRN protein in lignification. This study aimed to identify the function of Arabidopsis (Arabidopsis thaliana) PRN proteins in lignification of xylem tissues. Chemical composition of the secondary cell walls was analysed in Arabidopsis stems and/or hypocotyls by pyrolysis-gas chromatography/mass spectrometry, 2D-nuclear magnetic resonance and phenolic profiling. Secondary cell walls of individual xylem elements were chemotyped by Fourier transform infrared and Raman microspectroscopy. Arabidopsis PRN2 suppressed accumulation of S-type lignin in Arabidopsis stems and hypocotyls. PRN2 promoter activity and PRN2:GFP fusion protein were localised specifically in cells next to the vessel elements, suggesting a role for PRN2 in noncell-autonomous lignification of xylem vessels. Accordingly, PRN2 modulated lignin chemistry in the secondary cell walls of the neighbouring vessel elements. These results indicate that PRN2 suppresses S-type lignin accumulation in the neighbourhood of xylem vessels to bestow G-type enriched lignin composition on the secondary cell walls of the vessel elements. Gene expression analyses suggested that PRN2 function is mediated by regulation of the expression of the lignin-biosynthetic genes., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Coumarins, also known as 1,2-benzopyrones, comprise a large class of secondary metabolites that are ubiquitously found throughout the plant kingdom. In many plant species, coumarins are particularly important for iron acquisition and plant defence. Here, we show that COUMARIN SYNTHASE (COSY) is a key enzyme in the biosynthesis of coumarins. Arabidopsis thaliana cosy mutants have strongly reduced levels of coumarin and accumulate o-hydroxyphenylpropanoids instead. Accordingly, cosy mutants have reduced iron content and show growth defects when grown under conditions in which there is a limited availability of iron. Recombinant COSY is able to produce umbelliferone, esculetin and scopoletin from their respective o-hydroxycinnamoyl-CoA thioesters by two reaction steps-a trans-cis isomerization followed by a lactonization. This conversion happens partially spontaneously and is catalysed by light, which explains why the need for an enzyme for this conversion has been overlooked. The combined results show that COSY has an essential function in the biosynthesis of coumarins in organs that are shielded from light, such as roots. These findings provide routes to improving coumarin production in crops or by microbial fermentation.

Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinforces the cell walls, facilitates water transport, and acts as a physical barrier to pathogens. Lignin is typically described as being composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units that derive from the polymerization of the hydroxycinnamyl alcohols, p-coumaryl, coniferyl, and sinapyl alcohol, respectively. However, lignin also derives from various other aromatic monomers. Here, we review the biosynthetic pathway to the lignin monomers, and how flux through the pathway is regulated. Upon perturbation of the phenylpropanoid pathway, pathway intermediates may successfully incorporate into the lignin polymer, thereby affecting its physicochemical properties, or may remain soluble as such or as derivatized molecules that might interfere with physiological processes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Caffeic acid O-methyltransferase (COMT), the lignin biosynthesis gene modified in many brown-midrib high-digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl-to-guaiacyl ratio was reduced by ~50%, the 5-hydroxyguaiacyl (5-OH-G) unit incorporated into lignin at 10--15-fold higher levels than normal, and the amount of p-coumaric acid ester-linked to cell walls was reduced by ~50%. No brown-midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown-midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Lignin is the main cause of lignocellulosic biomass recalcitrance to industrial enzymatic hydrolysis. By partially replacing the traditional lignin monomers by alternative ones, lignin extractability can be enhanced. To design a lignin that is easier to degrade under alkaline conditions, curcumin (diferuloylmethane) was produced in the model plant Arabidopsis thaliana via simultaneous expression of the turmeric (Curcuma longa) genes DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE 2 (CURS2). The transgenic plants produced a plethora of curcumin- and phenylpentanoid-derived compounds with no negative impact on growth. Catalytic hydrogenolysis gave evidence that both curcumin and phenylpentanoids were incorporated into the lignifying cell wall, thereby significantly increasing saccharification efficiency after alkaline pretreatment of the transgenic lines by 14-24% as compared with the wild type. These results demonstrate that non-native monomers can be synthesized and incorporated into the lignin polymer in plants to enhance their biomass processing efficiency.

Background: Lignocellulosic biomass, such as wood and straw, is an interesting feedstock for the production of fermentable sugars. However, mainly due to the presence of lignin, this type of biomass is recalcitrant to saccharification. In Arabidopsis, lignocellulosic biomass with a lower lignin content or with lignin with an increased fraction of guaiacyl (G) and 5-hydroxyguaiacyl (5H) units shows an increased saccharification efficiency. Here, we stacked these two traits and studied the effect on the saccharification efficiency and biomass yield, by combining either transaldolase ( tra2 ), cinnamate 4 - hydroxylase ( c4h - 3 ), or 4 - coumarate:CoA ligase ( 4cl1 - 1 ) with caffeic acid O - methyltransferase ( comt - 1 or comt - 4 ) mutants., Results: The three double mutants ( tra2 comt - 1 , c4h - 3 comt - 4, and 4cl1 - 1 comt - 4 ) had a decreased lignin amount and an increase in G and 5H units in the lignin polymer compared to wild-type (WT) plants. The tra2 comt - 1 double mutant had a better saccharification efficiency compared to the parental lines when an acid or alkaline pretreatment was used. For the double mutants, c4h - 3 comt - 4 and 4cl1 - 1 comt - 4 , the saccharification efficiency was significantly higher compared to WT and its parental lines, independent of the pretreatment used. When no pretreatment was used, the saccharification efficiency increased even synergistically for these mutants., Conclusion: Our results show that saccharification efficiency can be improved by combining two different mutant lignin traits, leading to plants with an even higher saccharification efficiency, without having a yield reduction of the primary inflorescence stem. This approach can help improve saccharification efficiency in bio-energy crops.

Main Conclusion: CAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°-24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars due to the presence of lignin. To render lignocellulosic biomass a suitable feedstock for the bio-based economy, plants can be engineered to have decreased amounts of lignin. However, engineered plants with the lowest amounts of lignin exhibit collapsed vessels and yield penalties. Previous efforts were not able to fully overcome this phenotype without settling in sugar yield upon saccharification. Here, we reintroduced CINNAMOYL-COENZYME A REDUCTASE1 ( CCR1 ) expression specifically in the protoxylem and metaxylem vessel cells of Arabidopsis ( Arabidopsis thaliana ) ccr1 mutants. The resulting ccr1 ProSNBE : CCR1 lines had overcome the vascular collapse and had a total stem biomass yield that was increased up to 59% as compared with the wild type. Raman analysis showed that monolignols synthesized in the vessels also contribute to the lignification of neighboring xylary fibers. The cell wall composition and metabolome of ccr1 ProSNBE : CCR1 still exhibited many similarities to those of ccr1 mutants, regardless of their yield increase. In contrast to a recent report, the yield penalty of ccr1 mutants was not caused by ferulic acid accumulation but was (largely) the consequence of collapsed vessels. Finally, ccr1 ProSNBE : CCR1 plants had a 4-fold increase in total sugar yield when compared with wild-type plants., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar ( Populus tremula × Populus alba ) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 ( CAD1 ) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1 , coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S'(8-8)S' and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis ( Arabidopsis thaliana ) and later in Medicago truncatula However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar ( Populus tremula × Populus alba ) resulted in up to 25% reduced lignin deposition, increased levels of p -hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre-treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure-function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the β-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized β-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization., (© 2016 American Society of Plant Biologists. All rights reserved.)

Strigolactones are plant metabolites that act as phytohormones and rhizosphere signals. Whereas most research on unraveling the action mechanisms of strigolactones is focused on plant shoots, we investigated proteome adaptation during strigolactone signaling in the roots of Arabidopsis thaliana. Through large-scale, time-resolved, and quantitative proteomics, the impact of the strigolactone analog rac-GR24 was elucidated on the root proteome of the wild type and the signaling mutant more axillary growth 2 (max2). Our study revealed a clear MAX2-dependent rac-GR24 response: an increase in abundance of enzymes involved in flavonol biosynthesis, which was reduced in the max2-1 mutant. Mass spectrometry-driven metabolite profiling and thin-layer chromatography experiments demonstrated that these changes in protein expression lead to the accumulation of specific flavonols. Moreover, quantitative RT-PCR revealed that the flavonol-related protein expression profile was caused by rac-GR24-induced changes in transcript levels of the corresponding genes. This induction of flavonol production was shown to be activated by the two pure enantiomers that together make up rac-GR24. Finally, our data provide much needed clues concerning the multiple roles played by MAX2 in the roots and a comprehensive view of the rac-GR24-induced response in the root proteome., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)