Enhancing monolignol ferulate conjugate levels in poplar lignin via OsFMT1.

BACKGROUND: The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. RESULTS: Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV-Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic p oplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. CONCLUSIONS: The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.

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

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

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

FLA11 and FLA12 glycoproteins fine-tune stem secondary wall properties in response to mechanical stresses.

Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW-specific Fasciclin-Like Arabinogalactan-proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE-FLA11) showed earlier SCW development compared to the wild-type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE-FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE-FLA11, fla12, and OE-FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA-seq analysis revealed the up-regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE-FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine-tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins.

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Tailoring renewable materials via plant biotechnology.

Plants inherently display a rich diversity in cell wall chemistry, as they synthesize an array of polysaccharides along with lignin, a polyphenolic that can vary dramatically in subunit composition and interunit linkage complexity. These same cell wall chemical constituents play essential roles in our society, having been isolated by a variety of evolving industrial processes and employed in the production of an array of commodity products to which humans are reliant. However, these polymers are inherently synthesized and intricately packaged into complex structures that facilitate plant survival and adaptation to local biogeoclimatic regions and stresses, not for ease of deconstruction and commercial product development. Herein, we describe evolving techniques and strategies for altering the metabolic pathways related to plant cell wall biosynthesis, and highlight the resulting impact on chemistry, architecture, and polymer interactions. Furthermore, this review illustrates how these unique targeted cell wall modifications could significantly extend the number, diversity, and value of products generated in existing and emerging biorefineries. These modifications can further target the ability for processing of engineered wood into advanced high performance materials. In doing so, we attempt to illuminate the complex connection on how polymer chemistry and structure can be tailored to advance renewable material applications, using all the chemical constituents of plant-derived biopolymers, including pectins, hemicelluloses, cellulose, and lignins.

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar.

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.

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.

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.

Tailoring poplar lignin without yield penalty by combining a null and haploinsufficient CINNAMOYL-CoA REDUCTASE2 allele.

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.

Lignin Engineering in Forest Trees.

Wood is a renewable resource that is mainly composed of lignin and cell wall polysaccharides. The polysaccharide fraction is valuable as it can be converted into pulp and paper, or into fermentable sugars. On the other hand, the lignin fraction is increasingly being considered a valuable source of aromatic building blocks for the chemical industry. The presence of lignin in wood is one of the major recalcitrance factors in woody biomass processing, necessitating the need for harsh chemical treatments to degrade and extract it prior to the valorization of the cell wall polysaccharides, cellulose and hemicellulose. Over the past years, large research efforts have been devoted to engineering lignin amount and composition to reduce biomass recalcitrance toward chemical processing. We review the efforts made in forest trees, and compare results from greenhouse and field trials. Furthermore, we address the value and potential of CRISPR-based gene editing in lignin engineering and its integration in tree breeding programs.

Introducing curcumin biosynthesis in Arabidopsis enhances lignocellulosic biomass processing.

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.

Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana.

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.

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass.

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.