Biofuels for a sustainable future.

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

Bracing for Abscission.

The detachment of plant organs is highly choreographed, requiring the enzymatic dissolution of the middle lamella between cell layers at the base of the detaching organ. Now, Lee et al. demonstrate that abscission efficiency and plant health rely on the spatial confinement of enzymatic activity and mechanical features that ensure a smooth separation.

A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in Arabidopsis.

The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in Arabidopsis that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical "brace" to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating de novo specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.

Morphomechanical Innovation Drives Explosive Seed Dispersal.

How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is controlled at the cellular scale by asymmetric lignin deposition within endocarp b cells-a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation. VIDEO ABSTRACT.

Selective lignin arylation for biomass fractionation and benign bisphenols.

Lignocellulose is mainly composed of hydrophobic lignin and hydrophilic polysaccharide polymers, contributing to an indispensable carbon resource for green biorefineries1,2. When chemically treated, lignin is compromised owing to detrimental intra- and intermolecular crosslinking that hampers downstream process3,4. The current valorization paradigms aim to avoid the formation of new C-C bonds, referred to as condensation, by blocking or stabilizing the vulnerable moieties of lignin5-7. Although there have been efforts to enhance biomass utilization through the incorporation of phenolic additives8,9, exploiting lignin's proclivity towards condensation remains unproven for valorizing both lignin and carbohydrates to high-value products. Here we leverage the proclivity by directing the C-C bond formation in a catalytic arylation pathway using lignin-derived phenols with high nucleophilicity. The selectively condensed lignin, isolated in near-quantitative yields while preserving its prominent cleavable ß-ether units, can be unlocked in a tandem catalytic process involving aryl migration and transfer hydrogenation. Lignin in wood is thereby converted to benign bisphenols (34-48 wt%) that represent performance-advantaged replacements for their fossil-based counterparts. Delignified pulp from cellulose and xylose from xylan are co-produced for textile fibres and renewable chemicals. This condensation-driven strategy represents a key advancement complementary to other promising monophenol-oriented approaches targeting valuable platform chemicals and materials, thereby contributing to holistic biomass valorization.

Dearomatization drives complexity generation in freshwater organic matter.

Dissolved organic matter (DOM) is one of the most complex, dynamic and abundant sources of organic carbon, but its chemical reactivity remains uncertain1-3. Greater insights into DOM structural features could facilitate understanding its synthesis, turnover and processing in the global carbon cycle4,5. Here we use complementary multiplicity-edited 13C nuclear magnetic resonance (NMR) spectra to quantify key substructures assembling the carbon skeletons of DOM from four main Amazon rivers and two mid-size Swedish boreal lakes. We find that one type of reaction mechanism, oxidative dearomatization (ODA), widely used in organic synthetic chemistry to create natural product scaffolds6-10, is probably a key driver for generating structural diversity during processing of DOM that are rich in suitable polyphenolic precursor molecules. Our data suggest a high abundance of tetrahedral quaternary carbons bound to one oxygen and three carbon atoms (OCqC3 units). These units are rare in common biomolecules but could be readily produced by ODA of lignin-derived and tannin-derived polyphenols. Tautomerization of (poly)phenols by ODA creates non-planar cyclohexadienones, which are subject to immediate and parallel cycloadditions. This combination leads to a proliferation of structural diversity of DOM compounds from early stages of DOM processing, with an increase in oxygenated aliphatic structures. Overall, we propose that ODA is a key reaction mechanism for complexity acceleration in the processing of DOM molecules, creation of new oxygenated aliphatic molecules and that it could be prevalent in nature.

Bonding wood with uncondensed lignins as adhesives.

Plywood is widely used in construction, such as for flooring and interior walls, as well as in the manufacture of household items such as furniture and cabinets. Such items are made of wood veneers that are bonded together with adhesives such as urea-formaldehyde and phenol-formaldehyde resins1,2. Researchers in academia and industry have long aimed to synthesize lignin-phenol-formaldehyde resin adhesives using biomass-derived lignin, a phenolic polymer that can be used to substitute the petroleum-derived phenol3-6. However, lignin-phenol-formaldehyde resin adhesives are less attractive to plywood manufacturers than urea-formaldehyde and phenol-formaldehyde resins owing to their appearance and cost. Here we report a simple and practical strategy for preparing lignin-based wood adhesives from lignocellulosic biomass. Our strategy involves separation of uncondensed or slightly condensed lignins from biomass followed by direct application of a suspension of the lignin and water as an adhesive on wood veneers. Plywood products with superior performances could be prepared with such lignin adhesives at a wide range of hot-pressing temperatures, enabling the use of these adhesives as promising alternatives to traditional wood adhesives in different market segments. Mechanistic studies indicate that the adhesion mechanism of such lignin adhesives may involve softening of lignin by water, filling of vessels with softened lignin and crosslinking of lignins in adhesives with those in the cell wall.

A mechanism for localized lignin deposition in the endodermis.

The precise localization of extracellular matrix and cell wall components is of critical importance for multicellular organisms. Lignin is a major cell wall modification that often forms intricate subcellular patterns that are central to cellular function. Yet the mechanisms of lignin polymerization and the subcellular precision of its formation remain enigmatic. Here, we show that the Casparian strip, a lignin-based, paracellular diffusion barrier in plants, forms as a precise, median ring by the concerted action of a specific, localized NADPH oxidase, brought into proximity of localized peroxidases through the action of Casparian strip domain proteins (CASPs). Our findings in Arabidopsis provide a simple mechanistic model of how plant cells regulate lignin formation with subcellular precision. We speculate that scaffolding of NADPH oxidases to the downstream targets of the reactive oxygen species (ROS) that they produce might be a widespread mechanism to ensure specificity and subcellular precision of ROS action within the extracellular matrix.

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.

Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis.

Vascular plants reinforce the cell walls of the different xylem cell types with lignin phenolic polymers. Distinct lignin chemistries differ between each cell wall layer and each cell type to support their specific functions. Yet the mechanisms controlling the tight spatial localization of specific lignin chemistries remain unclear. Current hypotheses focus on control by monomer biosynthesis and/or export, while cell wall polymerization is viewed as random and nonlimiting. Here, we show that combinations of multiple individual laccases (LACs) are nonredundantly and specifically required to set the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12, and 17 by generating quadruple and quintuple loss-of-function mutants. Loss of these LACs in different combinations led to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. Moreover, we showed that LAC-mediated lignification has distinct functions in specific cell types, waterproofing fibers, and strengthening vessels. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with nonredundant activities immobilized in specific cell types and cell wall layers.

Gibberellin signaling regulates lignin biosynthesis to modulate rice seed shattering.

The elimination of seed shattering was a key step in rice (Oryza sativa) domestication. In this paper, we show that increasing the gibberellic acid (GA) content or response in the abscission region enhanced seed shattering in rice. We demonstrate that SLENDER RICE1 (SLR1), the key repressor of GA signaling, could physically interact with the rice seed shattering-related transcription factors quantitative trait locus of seed shattering on chromosome 1 (qSH1), O. sativa HOMEOBOX 15 (OSH15), and SUPERNUMERARY BRACT (SNB). Importantly, these physical interactions interfered with the direct binding of these three regulators to the lignin biosynthesis gene 4-COUMARATE: COENZYME A LIGASE 3 (4CL3), thereby derepressing its expression. Derepression of 4CL3 led to increased lignin deposition in the abscission region, causing reduced rice seed shattering. Importantly, we also show that modulating GA content could alter the degree of seed shattering to increase harvest efficiency. Our results reveal that the "Green Revolution" phytohormone GA is important for regulating rice seed shattering, and we provide an applicable breeding strategy for high-efficiency rice harvesting.

The nuclear effector ArPEC25 from the necrotrophic fungus Ascochyta rabiei targets the chickpea transcription factor CaßLIM1a and negatively modulates lignin biosynthesis, increasing host susceptibility.

Fungal pathogens deploy a barrage of secreted effectors to subvert host immunity, often by evading, disrupting, or altering key components of transcription, defense signaling, and metabolic pathways. However, the underlying mechanisms of effectors and their host targets are largely unexplored in necrotrophic fungal pathogens. Here, we describe the effector protein Ascochyta rabiei PEXEL-like Effector Candidate 25 (ArPEC25), which is secreted by the necrotroph A. rabiei, the causal agent of Ascochyta blight disease in chickpea (Cicer arietinum), and is indispensable for virulence. After entering host cells, ArPEC25 localizes to the nucleus and targets the host LIM transcription factor CaßLIM1a. CaßLIM1a is a transcriptional regulator of CaPAL1, which encodes phenylalanine ammonia lyase (PAL), the regulatory, gatekeeping enzyme of the phenylpropanoid pathway. ArPEC25 inhibits the transactivation of CaßLIM1a by interfering with its DNA-binding ability, resulting in negative regulation of the phenylpropanoid pathway and decreased levels of intermediates of lignin biosynthesis, thereby suppressing lignin production. Our findings illustrate the role of fungal effectors in enhancing virulence by targeting a key defense pathway that leads to the biosynthesis of various secondary metabolites and antifungal compounds. This study provides a template for the study of less explored necrotrophic effectors and their host target functions.

Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE.

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. The reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

Evolution of sea-surfing plant propagule as revealed by the genomes of Heritiera mangroves.

Coastal forests, such as mangroves, protect much of the tropical and subtropical coasts. Long-distance dispersal via sea-surfing propagules is essential for coastal plants, but the genomic and molecular basis of sea-surfing plant propagule evolution remains unclear. Heritiera fomes and Heritiera littoralis are two coastal plants with typical buoyant fruits. We de novo sequenced and assembled their high-quality genomes. Our phylogenomic analysis indicates H. littoralis and H. fomes originated (at ~6.08 Mya) just before the start of Quaternary sea-level fluctuations. Whole-genome duplication occurred earlier, permitting gene copy gains in the two species. Many of the expanded gene families are involved in lignin and flavonoid biosynthesis, likely contributing to buoyant fruit emergence. It is repeatedly revealed that one duplicated copy to be under positive selection while the other is not. By examining H. littoralis fruits at three different developmental stages, we found that gene expression levels remain stable from young to intermediate. However, ~1000 genes are up-regulated and ~ 3000 genes are down-regulated as moving to mature. Particularly in fruit epicarps, the upregulation of WRKY12 and E2Fc likely constrains the production of p-Coumaroyl-CoA, the key internal substrate for lignin biosynthesis. Hence, to increase fruit impermeability, methylated lignin biosynthesis is shut down by down-regulating the genes CCoAOMT, F5H, COMT, and CSE, while unmethylated lignins are preferentially produced by upregulating CAD and CCR. Similarly, cutin polymers and cuticular waxes accumulate with high levels before maturation in epicarps. Overall, our genome assemblies and analyses uncovered the genomic evolution and temporal transcriptional regulation of sea-surfing propagule.

CsPAT1, a GRAS transcription factor, promotes lignin accumulation by antagonistic interacting with CsWRKY13 in tea plants.

Lignin is an important component of plant cell walls and plays crucial roles in the essential agronomic traits of tea quality and tenderness. However, the molecular mechanisms underlying the regulation of lignin biosynthesis in tea plants remain unclear. CsWRKY13 acts as a negative regulator of lignin biosynthesis in tea plants. In this study, we identified a GRAS transcription factor, phytochrome A signal transduction 1 (CsPAT1), that interacts with CsWRKY13. Silencing CsPAT1 expression in tea plants and heterologous overexpression in Arabidopsis demonstrated that CsPAT1 positively regulates lignin accumulation. Further investigation revealed that CsWRKY13 directly binds to the promoters of CsPAL and CsC4H and suppresses transcription of CsPAL and CsC4H. CsPAT1 indirectly affects the promoter activities of CsPAL and CsC4H by interacting with CsWRKY13, thereby facilitating lignin biosynthesis in tea plants. Compared with the expression of CsWRKY13 alone, the co-expression of CsPAT1 and CsWRKY13 in Oryza sativa significantly increased lignin biosynthesis. Conversely, compared with the expression of CsPAT1 alone, the co-expression of CsPAT1 and CsWRKY13 in O. sativa significantly reduced lignin accumulation. These results demonstrated the antagonistic regulation of the lignin biosynthesis pathway by CsPAT1 and CsWRKY13. These findings improve our understanding of lignin biosynthesis mechanisms in tea plants and provide insights into the role of the GRAS transcription factor family in lignin accumulation.

Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis.

Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.

Metabolic adaptations leading to an enhanced lignification in wheat roots under salinity stress.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Characterization of the degradation products of lignocellulosic biomass by using tandem mass spectrometry experiments, model compounds, and quantum chemical calculations.

Biomass-derived degraded lignin and cellulose serve as possible alternatives to fossil fuels for energy and chemical resources. Fast pyrolysis of lignocellulosic biomass generates bio-oil that needs further refinement. However, as pyrolysis causes massive degradation to lignin and cellulose, this process produces very complex mixtures. The same applies to degradation methods other than fast pyrolysis. The ability to identify the degradation products of lignocellulosic biomass is of great importance to be able to optimize methodologies for the conversion of these mixtures to transportation fuels and valuable chemicals. Studies utilizing tandem mass spectrometry have provided invaluable, molecular-level information regarding the identities of compounds in degraded biomass. This review focuses on the molecular-level characterization of fast pyrolysis and other degradation products of lignin and cellulose via tandem mass spectrometry based on collision-activated dissociation (CAD). Many studies discussed here used model compounds to better understand both the ionization chemistry of the degradation products of lignin and cellulose and their ions' CAD reactions in mass spectrometers to develop methods for the structural characterization of the degradation products of lignocellulosic biomass. Further, model compound studies were also carried out to delineate the mechanisms of the fast pyrolysis reactions of lignocellulosic biomass. The above knowledge was used to assign likely structures to many degradation products of lignocellulosic biomass.