The mode of transport of lignin monomers to the sites of polymerization in the apoplast remains controversial. C-Lignin is a recently discovered form of lignin found in some seed coats that is composed exclusively of units derived from caffeyl alcohol. RNA-seq and proteome analyses identified a number of transporters co-expressed with C-lignin deposition in the seed coat of Cleome hassleriana. Cloning and influx/efflux analysis assays in yeast identified two low-affinity transporters, ChPLT3 and ChSUC1, that were active with caffeyl alcohol but not with the classical monolignols p-coumaryl, coniferyl, and sinapyl alcohols, consistent with molecular modeling and docking studies. Expression of ChPLT3 in Arabidopsis seedlings enhanced root growth in the presence of caffeyl alcohol, and expression of ChPLT3 and ChSUC1 correlated with lignin C-unit content in hairy roots of Medicago truncatula. We present a model, consistent with phylogenetic and evolutionary considerations, whereby passive caffeyl alcohol transport may be supplemented by hitchhiking on secondary active transporters to ensure the synthesis of C-lignin, and inhibition of synthesis of G-lignin, in the apoplast.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

The bioconversion of homogeneous linear catechyl lignin (C-lignin) to polyhydroxyalkanoates (PHA) was examined for the first time in this study. C-lignins from vanilla, euphorbia, and candlenut seed coats (denoted as C1, C2, and C3, respectively) varied in their molecular structures, which showed different molecular weight distributions, etherification degrees, and contents of hydroxyl groups. A notable amount of nonetherified catechol units existed within C1 and C2 lignins, and these catechol units were consumed during fermentation. These results suggested that the nonetherified catechol structure was readily converted by Pseudomonas putida KT2440. Since the weight-average molecular weight of C2 raw lignin was 26.7% lower than that of C1, the bioconversion performance of C2 lignin was more outstanding. The P. putida KT2440 cell amount reached the maximum of 9.3 × 107 CFU/mL in the C2 medium, which was 37.9 and 82.4% higher than that in the C1 and C3 medium, respectively. Accordingly, PHA concentration reached 137 mg/L within the C2 medium, which was 41.2 and 149.1% higher than the C1 and C3 medium, respectively. Overall, C-lignin, with a nonetherified catechol structure and low molecular weight, benefits its microbial conversion significantly.

The aerial roots of vanilla plant contain an strikingly high level of tricin in lignin (29.1 mg g −1 ). It will be a noteworthy feedstock for value-added chemicals and commodities to advance plant bioengineering and lignin valorization.

The factors controlling lignin composition remain unclear. Catechyl (C)-lignin is a homopolymer of caffeyl alcohol with unique properties as a biomaterial and precursor of industrial chemicals. The lignin synthesized in the seed coat of Cleome hassleriana switches from guaiacyl (G)- to C-lignin at around 12 to 14 days after pollination (DAP), associated with a rerouting of the monolignol pathway. Lack of synthesis of caffeyl alcohol limits C-lignin formation before around 12 DAP, but coniferyl alcohol is still synthesized and highly accumulated after 14 DAP. We propose a model in which, during C-lignin biosynthesis, caffeyl alcohol noncompetitively inhibits oxidation of coniferyl alcohol by cell wall laccases, a process that might limit movement of coniferyl alcohol to the apoplast. Developmental changes in both substrate availability and laccase specificity together account for the metabolic fates of G- and C-monolignols in the Cleome seed coat.

Lignin is a complex phenolic polymer that imparts cell wall strength, facilitates water transport and functions as a physical barrier to pathogens in all vascular plants. Lignin biosynthesis is a carbon-consuming, non-reversible process, which requires tight regulation. Here, we report that a major monomer unit of the lignin polymer can function as a signal molecule to trigger proteolysis of the enzyme L-phenylalanine ammonia-lyase, the entry point into the lignin biosynthetic pathway, and feedback regulate the expression levels of lignin biosynthetic genes. These findings highlight the highly complex regulation of lignin biosynthesis and shed light on the biological importance of monolignols as signaling molecules.

Catechyl lignin (C-lignin) is a linear homopolymer of caffeyl alcohol found in the seed coats of diverse plant species. Its properties make it a natural source of carbon fibers and high-value chemicals, but the mechanism of in planta polymerization of caffeyl alcohol remains unclear. In the ornamental plant Cleome hassleriana, lignin biosynthesis in the seed coat switches from guaiacyl lignin to C-lignin at ∼12 d after pollination. Here we found that the transcript profile of the laccase gene ChLAC8 parallels the accumulation of C-lignin during seed coat development. Recombinant ChLAC8 oxidizes caffeyl and sinapyl alcohols, generating their corresponding dimers or trimers in vitro, but cannot oxidize coniferyl alcohol. We propose a basis for this substrate preference based on molecular modeling/docking experiments. Suppression of ChLAC8 expression led to significantly reduced C-lignin content in the seed coats of transgenic Cleome plants. Feeding of 13C-caffeyl alcohol to the Arabidopsis (Arabidopsis thaliana) caffeic acid o-methyltransferase mutant resulted in no incorporation of 13C into C-lignin, but expressing ChLAC8 in this genetic background led to appearance of C-lignin with >40% label incorporation. These results indicate that ChLAC8 is required for C-lignin polymerization and determines lignin composition when caffeyl alcohol is available.

C-lignin is a linear polymer of caffeyl alcohol, found in the seed coats of several exotic plant species, with promising properties for generation of carbon fibers and high value chemicals. In the ornamental plant Cleome hassleriana, guaiacyl (G) lignin is deposited in the seed coat for the first 6-12 days after pollination, after which G-lignin deposition ceases and C-lignin accumulates, providing an excellent model system to study C-lignin biosynthesis. We performed RNA sequencing of seed coats harvested at 2-day intervals throughout development. Bioinformatic analysis identified a complete set of lignin biosynthesis genes for Cleome. Transcript analysis coupled with kinetic analysis of recombinant enzymes in Escherichia coli revealed that the switch to C-lignin formation was accompanied by down-regulation of transcripts encoding functional caffeoyl CoA- and caffeic acid 3-O-methyltransferases (CCoAOMT and COMT) and a form of cinnamyl alcohol dehydrogenase (ChCAD4) with preference for coniferaldehyde as substrate, and up-regulation of a form of CAD (ChCAD5) with preference for caffealdehyde. Based on these analyses, blockage of lignin monomer methylation by down-regulation of both O-methyltransferases (OMTs) and methionine synthase (for provision of C1 units) appears to be the major factor in diversion of flux to C-lignin in the Cleome seed coat, although the change in CAD specificity also contributes based on the reduction of C-lignin levels in transgenic Cleome with down-regulation of ChCAD5. Structure modeling and mutational analysis identified amino acid residues important for the preference of ChCAD5 for caffealdehyde.

Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development but is a major impediment to efficient biomass utilization in various processes. Valorizing lignin is increasingly realized as being essential. However, rapid condensation of lignin during acidic extraction leads to the formation of recalcitrant condensed units that, along with similar units and structural heterogeneity in native lignin, drastically limits product yield and selectivity. Catechyl lignin (C-lignin), which is essentially a benzodioxane homopolymer without condensed units, might represent an ideal lignin for valorization, as it circumvents these issues. We discovered that C-lignin is highly acid-resistant. Hydrogenolysis of C-lignin resulted in the cleavage of all benzodioxane structures to produce catechyl-type monomers in near-quantitative yield with a selectivity of 90% to a single monomer.

Comunicacion oral presentada en la XXIX Conferencia Internacional sobre Polifenoles y la 9a Conferencia de Taninos, del 16 al 20 de julio de 2018 Madison, WI, USA

Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co-expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co-expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down-regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TFs in controlling secondary wall formation. RNA-seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall-related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta-analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

BACKGROUND:Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass (Panicum virgatum) have yet to be reported.RESULTS:Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation.CONCLUSION:Together, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.

A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a “vanillin synthase” could enable biotechnological production of vanillin from ferulic acid using a “natural” vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like “vanillin synthase” that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined.

Background: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. Results: A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. Conclusions: A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, largescale, multi-institutional reverse genetic pipelines to improve crop traits.

L-Phenylalanine ammonia-lyase (PAL) is the first enzyme in the biosynthesis of phenylpropanoid-derived plant compounds such as flavonoids, coumarins and the cell wall polymer lignin. The cell walls of grasses possess higher proportions of syringyl (S)-rich lignins and high levels of esterified coumaric acid compared with those of dicotyledonous plants, and PAL from grasses can also possess tyrosine ammonia-lyase (TAL) activity, the reason for which has remained unclear. Using phylogenetic, transcriptomic and in vitro biochemical analyses, we identified a single homotetrameric bifunctional ammonia-lyase (PTAL) among eight BdPAL enzymes in the model grass species Brachypodium distachyon. (13)C isotope labelling experiments along with BdPTAL1-downregulation in transgenic plants showed that the TAL activity of BdPTAL1 can provide nearly half of the total lignin deposited in Brachypodium, with a preference for S-lignin and wall-bound coumarate biosynthesis, indicating that PTAL function is linked to the characteristic features of grass cell walls. Furthermore, isotope dilution experiments suggest that the pathways to lignin from L-phenylalanine and L-tyrosine are distinct beyond the formation of 4-coumarate, supporting the organization of lignin synthesis enzymes in one or more metabolons.

Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.

通过进行实验室小试实验,研究了生物滤池对7种农药的去除效果.结果表明,生物滤池对7种农药三唑酮、扑草净、草净津、敌草隆、扑灭津、莠去津、仲丁威均表现出一定的去除率.其中三唑酮、扑草净、草净津、扑灭津、莠去津、仲丁威的去除主要是通过微生物的分解代谢和共代谢作用,而敌草隆难以生物降解.本研究表明,葡萄糖对生物降解三唑酮、扑草净、草净津、扑灭津、莠去津、仲丁威有促进作用,氨氮对农药的降解无明显影响.沿水流方向,7种农药含量基本保持沿程递减的趋势,其去除主要集中在滤层深度0～50 cm范围内.

High biomass yields and minimal agronomic input requirements have made switchgrass, Panicum virgatum L., a leading candidate lignocellulosic bioenergy crop. Large-scale lignocellulosic biofuel production from such crops is limited by the difficulty to deconstruct cell walls into fermentable sugars: the recalcitrance problem. Our goal in this study was to assess the field performance of switchgrass plants overexpressing the switchgrass MYB4 (PvMYB4) transcription factor gene. PvMYB4 transgenic switchgrass can have great lignin reduction, which commensurately increases sugar release and biofuel production. Our results over two growing seasons showed that one transgenic event (out of eight) had important gains in both biofuel (32 % more) and biomass (63 % more) at the end of the second growing season relative to non-transgenic controls. These gains represent a doubling of biofuel production per hectare, which is the highest gain reported from any field-grown modified feedstock. In contrast to this transgenic event, which had relatively low ectopic overexpression of the transgene, five of the eight transgenic events planted did not survive the first field winter. The dead plants were all high-overexpressing events that performed well in the earlier greenhouse studies. Disease susceptibility was not compromised in any transgenic events over the field experiments. These results demonstrate the power of modifying the expression of an endogenous transcription factor to improve biofuel and biomass simultaneously, and also highlight the importance of field studies for "sorting" transgenic events. Further research is needed to develop strategies for fine-tuning temporal-spatial transgene expression in feedstocks to optimize desired phenotypes.

BACKGROUND:One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants.RESULTS:Consistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis.CONCLUSIONS:Our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in above ground biomass, selective down-regulation of individual components of C1 metabolism is an approach that should be explored further for the improvement of lignocellulosic feedstocks.

Lignocellulosic biomass has the potential to be a significant source of renewable energy, provided that current challenges regarding sufficient biomass production and efficient biomass conversion are met. Floral transition is a major developmental switch in the life of flowering plants which dictates whether photosynthetic products should be invested in vegetative growth or reproductive development. To directly evaluate the contribution of floral transition to biomass yield, we manipulated the onset of flowering in the model legume Medicago truncatula using a mutagenesis approach. Through forward genetics screening of Tnt1-tagged mutants, we identified three M. truncatula lines altered in flowering time and fertility: a delayed flowering mutant named vernalization-insensitive delayed flowering in long days (vdf), a nonflowering stemless mutant named headless (hdl), and a male sterile mutant named medicago male sterile 1 (mms1). Biomass yield analyses at different stages of development in these mutants revealed that the vdf mutant had the highest aboveground biomass and the hdl mutant had the lowest biomass at 70 days after germination. The difference in biomass yield between the vdf mutant and wild-type R108 became apparent after floral initiation and peaked at 90 days after germination, the late blooming stage for R108, where the vdf plants produced approximately twofold more biomass than R108. Interestingly, vdf, hdl, and mms1 mutants produced significantly less lignin than R108. Our results suggest that delaying floral initiation could be employed as a convenient tool to simultaneously improve biomass quantity and quality, provided that this is achieved without pleiotropic developmental defects.