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Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. The reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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.)

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extended previous resonance excitation-type collision-induced dissociation (CID) methods that identified lignin oligomers containing β-O-4, β-5, and β-β bonds, to also identify characteristics of 5-5, β-1, and 4-O-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, QTOF-ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification., (© 2022 Wiley-VCH GmbH.)

Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favorable net CO 2 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 virus 5 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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. 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.

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., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The plant specialized metabolome consists of a multitude of structurally and functionally diverse metabolites, variable from species to species. The specialized metabolites play roles in the response to environmental changes and abiotic or biotic stresses, as well as in plant growth and development. At its basis, the specialized metabolism is built of four major pathways, each starting from a few distinct primary metabolism precursors, and leading to distinct basic carbon skeleton core structures: polyketides and fatty acid derivatives, terpenoids, alkaloids, and phenolics. Structural diversity in specialized metabolism, however, expands exponentially with each subsequent modification. We review here the major sources of structural variety and question if a specific role can be attributed to each distinct structure. We focus on the influences that various core structures and modifications have on flavonoid antioxidant activity and on the diversity generated by oxidative coupling reactions. We suggest that many oxidative coupling products, triggered by initial radical scavenging, may not have a function in se , but could potentially be enzymatically recycled to effective antioxidants. We further discuss the wide structural variety created by multiple decorations (glycosylations, acylations, prenylations), the formation of high-molecular weight conjugates and polyesters, and the plasticity of the specialized metabolism. We draw attention to the need for untargeted methods to identify the complex, multiply decorated and conjugated compounds, in order to study the functioning of the plant specialized metabolome.

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.)

Lignin is a complex polymer deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

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).)

Over the last decade, a giant leap forward has been made in resolving the main bottleneck in metabolomics, i.e., the structural characterization of the many unknowns. This has led to the next challenge in this research field: retrieving biochemical pathway information from the various types of networks that can be constructed from metabolome data. Searching putative biochemical pathways, referred to as biotransformation paths, is complicated because several flaws occur during the construction of metabolome networks. Multiple network analysis tools have been developed to deal with these flaws, while in silico retrosynthesis is appearing as an alternative approach. In this review, the different types of metabolome networks, their flaws, and the various tools to trace these biotransformation paths are discussed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Hirschmanniella oryzae is one of the most devastating nematodes on rice, leading to substantial yield losses. Effector proteins aid the nematode during the infection process by subduing plant defence responses. In this research we characterized two potential H. oryzae effector proteins, chorismate mutase (HoCM) and isochorismatase (HoICM), and investigated their enzymatic activity and their role in plant immunity. Both HoCM and HoICM proved to be enzymatically active in complementation tests in mutant Escherichia coli strains. Infection success by the migratory nematode H. oryzae was significantly higher in transgenic rice lines constitutively expressing HoCM or HoICM. Expression of HoCM, but not HoICM, increased rice susceptibility against the sedentary nematode Meloidogyne graminicola also. Transcriptome and metabolome analyses indicated reductions in secondary metabolites in the transgenic rice plants expressing the potential nematode effectors. The results presented here demonstrate that both HoCM and HoICM suppress the host immune system and that this may be accomplished by lowering secondary metabolite levels in the plant., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

This study investigates the impact of the alteration of the monolignol biosynthesis pathway on the establishment of the in vitro interaction of poplar roots either with a mutualistic ectomycorrhizal fungus or with a pathogenic root-knot nematode. Overall, the five studied transgenic lines downregulated for caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) or both COMT and CAD displayed a lower mycorrhizal colonisation percentage, indicating a lower ability for establishing mutualistic interaction than the wild-type. The susceptibility to root-knot nematode infection was variable in the five lines, and the CAD-deficient line was found to be less susceptible than the wild-type. We discuss these phenotypic differences in the light of the large shifts in the metabolic profile and gene expression pattern occurring between roots of the CAD-deficient line and wild-type. A role of genes related to trehalose metabolism, phytohormones, and cell wall construction in the different mycorrhizal symbiosis efficiency and nematode sensitivity between these two lines is suggested. Overall, these results show that the alteration of plant metabolism caused by the repression of a single gene within phenylpropanoid pathway results in significant alterations, at the root level, in the response towards mutualistic and pathogenic associates. These changes may constrain plant fitness and biomass production, which are of economic importance for perennial industrial crops such as poplar.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity., (© 2020 John Wiley & Sons Ltd.)

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita , a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

Background: Cassava whitefly outbreaks were initially reported in East and Central Africa cassava (Manihot esculenta Crantz) growing regions in the 1990's and have now spread to other geographical locations, becoming a global pest severely affecting farmers and smallholder income. Whiteflies impact plant yield via feeding and vectoring cassava mosaic and brown streak viruses, making roots unsuitable for food or trading. Deployment of virus resistant varieties has had little impact on whitefly populations and therefore development of whitefly resistant varieties is also necessary as part of integrated pest management strategies. Suitable sources of whitefly resistance exist in germplasm collections that require further characterization to facilitate and assist breeding programs., Results: In the present work, a hierarchical metabolomics approach has been employed to investigate the underlying biochemical mechanisms associated with whitefly resistance by comparing two naturally occurring accessions of cassava, one susceptible and one resistant to whitefly. Quantitative differences between genotypes detected at pre-infestation stages were consistently observed at each time point throughout the course of the whitefly infestation. This prevalent differential feature suggests that inherent genotypic differences override the response induced by the presence of whitefly and that they are directly linked with the phenotype observed. The most significant quantitative changes relating to whitefly susceptibility were linked to the phenylpropanoid super-pathway and its linked sub-pathways: monolignol, flavonoid and lignan biosynthesis. These findings suggest that the lignification process in the susceptible variety is less active, as the susceptible accession deposits less lignin and accumulates monolignol intermediates and derivatives thereof, differences that are maintained during the time-course of the infestation., Conclusions: Resistance mechanism associated to the cassava whitefly-resistant accession ECU72 is an antixenosis strategy based on reinforcement of cell walls. Both resistant and susceptible accessions respond differently to whitefly attack at biochemical level, but the inherent metabolic differences are directly linked to the resistance phenotype rather than an induced response in the plant.

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.

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.)

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13 C 6 -glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts., (Copyright © 2017 Elsevier Ltd. 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.)

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H 2 O 2 ) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce ( Picea abies ) was used as a research model. Scavenging of apoplastic H 2 O 2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H 2 O 2 -scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H 2 O 2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H 2 O 2 production in addition to potential H 2 O 2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

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.)

Background: Caffeoyl shikimate esterase (CSE) was recently characterized as an enzyme central to the lignin biosynthetic pathway in Arabidopsis thaliana. The cse-2 loss-of-function mutant shows a typical phenotype of lignin-deficient mutants, including collapsed vessels, reduced lignin content, and lignin compositional shift, in addition to a fourfold increase in cellulose-to-glucose conversion when compared to the wild type. However, this mutant exhibits a substantial developmental arrest, which might outweigh the gains in fermentable sugar yield. To restore its normal growth and further improve its saccharification yield, we investigated a possible cause for the yield penalty of the cse-2 mutant. Furthermore, we evaluated whether CSE expression is under the same multi-leveled transcriptional regulatory network as other lignin biosynthetic genes and analyzed the transcriptional responses of the phenylpropanoid pathway upon disruption of CSE., Results: Transactivation analysis demonstrated that only second-level MYB master switches (MYB46 and MYB83) and lignin-specific activators (MYB63 and MYB85), but not top-level NAC master switches or other downstream transcription factors, effectively activate the CSE promoter in our protoplast-based system. The cse-2 mutant exhibited transcriptional repression of genes upstream of CSE, while downstream genes were mainly unaffected, indicating transcriptional feedback of CSE loss-of-function on monolignol biosynthetic genes. In addition, we found that the expression of CSE under the control of the vessel-specific VND7 promoter in the cse-2 background restored the vasculature integrity resulting in improved growth parameters, while the overall lignin content remained relatively low. Thus, by restoring the vascular integrity and biomass parameters of cse-2, we further improved glucose release per plant without pretreatment, with an increase of up to 36 % compared to the cse-2 mutant and up to 154 % compared to the wild type., Conclusions: Our results contribute to a better understanding of how the expression of CSE is regulated by secondary wall-associated transcription factors and how the expression of lignin genes is affected upon CSE loss-of-function in Arabidopsis. Moreover, we found evidence that vasculature collapse is underlying the yield penalty found in the cse-2 mutant. Through a vessel-specific complementation approach, vasculature morphology and final stem weight were restored, leading to an even higher total glucose release per plant.

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram-negative bacterium Sphingobium sp. SYK-6 possesses a Cα-dehydrogenase (LigD) enzyme that has been shown to oxidize the α-hydroxy functionalities in β-O-4-linked dimers into α-keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β-O-4-linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol-β-coniferyl alcohol ether and syringylglycerol-β-sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon-optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC-MS/MS-based metabolite profiling indicated that levels of oxidized guaiacyl (G) β-O-4-coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1- to 2.8-fold increased level of G-type α-keto-β-O-4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) straw and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4'-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4'-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4'-O-β)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in the cell wall. Recently, various glycosylated lignin oligomers have been revealed in Arabidopsis thaliana. Given that monolignol oxidation and monolignol radical coupling are known to occur in the apoplast, and glycosylation in the cytoplasm, it raises questions about the subcellular localization of glycosylated lignin oligomer biosynthesis and their storage. By metabolite profiling of Arabidopsis leaf vacuoles, we show that the leaf vacuole stores a large number of these small glycosylated lignin oligomers. Their structural variety and the incorporation of alternative monomers, as observed in Arabidopsis mutants with altered monolignol biosynthesis, indicate that they are all formed by combinatorial radical coupling. In contrast to the common believe that combinatorial coupling is restricted to the apoplast, we hypothesized that the aglycones of these compounds are made within the cell. To investigate this, leaf protoplast cultures were cofed with 13C6-labeled coniferyl alcohol and a 13C4-labeled dimer of coniferyl alcohol. Metabolite profiling of the cofed protoplasts provided strong support for the occurrence of intracellular monolignol coupling. We therefore propose a metabolic pathway involving intracellular combinatorial coupling of monolignol radicals, followed by oligomer glycosylation and vacuolar import, which shares characteristics with both lignin and lignan biosynthesis., (© 2015 American Society of Plant Biologists. All rights reserved.)

The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By performing a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv′/Fm′) was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family, which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signalling in stress tolerance.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply., (© 2014 American Society of Plant Biologists. All rights reserved.)

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta., (© 2014 American Society of Plant Biologists. All rights reserved.)

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane.

Plant metabolomics is increasingly used for pathway discovery and to elucidate gene function. However, the main bottleneck is the identification of the detected compounds. This is more pronounced for secondary metabolites as many of their pathways are still underexplored. Here, an algorithm is presented in which liquid chromatography-mass spectrometry profiles are searched for pairs of peaks that have mass and retention time differences corresponding with those of substrates and products from well-known enzymatic reactions. Concatenating the latter peak pairs, called candidate substrate-product pairs (CSPP), into a network displays tentative (bio)synthetic routes. Starting from known peaks, propagating the network along these routes allows the characterization of adjacent peaks leading to their structure prediction. As a proof-of-principle, this high-throughput cheminformatics procedure was applied to the Arabidopsis thaliana leaf metabolome where it allowed the characterization of the structures of 60% of the profiled compounds. Moreover, based on searches in the Chemical Abstract Service database, the algorithm led to the characterization of 61 compounds that had never been described in plants before. The CSPP-based annotation was confirmed by independent MS(n) experiments. In addition to being high throughput, this method allows the annotation of low-abundance compounds that are otherwise not amenable to isolation and purification. This method will greatly advance the value of metabolomics in systems biology.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

Next-generation (NG) sequencing in a natural population of Populus nigra revealed a mutant with a premature stop codon in the gene encoding hydroxycinnamoyl-CoA : shikimate hydroxycinnamoyl transferase1 (HCT1), an essential enzyme in lignin biosynthesis. The lignin composition of P. nigra trees homozygous for the defective allele was compared with that of heterozygous trees and trees without the defective allele. The lignin was characterized by phenolic profiling, lignin oligomer sequencing, thioacidolysis and NMR. In addition, HCT1 was heterologously expressed for activity assays and crosses were made to introduce the mutation in different genetic backgrounds. HCT1 converts p-coumaroyl-CoA into p-coumaroyl shikimate. The mutant allele, PnHCT1-Δ73, encodes a truncated protein, and trees homozygous for this recessive allele have a modified lignin composition characterized by a 17-fold increase in p-hydroxyphenyl units. Using the lignin pathway as proof of concept, we illustrated that the capture of rare defective alleles is a straightforward approach to initiate reverse genetics and accelerate tree breeding. The proposed breeding strategy, called 'breeding with rare defective alleles' (BRDA), should be widely applicable, independent of the target gene or the species., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

Lignin, a phenolic polymer in the secondary wall, is the major cause of lignocellulosic biomass recalcitrance to efficient industrial processing. From an applications perspective, it is desirable that second-generation bioenergy crops have lignin that is readily degraded by chemical pretreatments but still fulfill its biological role in plants. Because plants can tolerate large variations in lignin composition, often without apparent adverse effects, substitution of some fraction of the traditional monolignols by alternative monomers through genetic engineering is a promising strategy to tailor lignin in bioenergy crops. However, successful engineering of lignin incorporating alternative monomers requires knowledge about phenolic metabolism in plants and about the coupling properties of these alternative monomers. Here, we review the current knowledge about lignin biosynthesis and the pathways towards the main phenolic classes. In addition, the minimal requirements are defined for molecules that, upon incorporation into the lignin polymer, make the latter more susceptible to biomass pretreatment. Numerous metabolites made by plants meet these requirements, and several have already been tested as monolignol substitutes in biomimetic systems. Finally, the status of detection and identification of compounds by phenolic profiling is discussed, as phenolic profiling serves in pathway elucidation and for the detection of incorporation of alternative lignin monomers., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)