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Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable–properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

Background Horehound ( Marrubium vulgare ) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, Mv CPS1 and Mv ELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. Results Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential Mv CPS1 and Mv ELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta , high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare . Conclusions In a sequential reaction with the diterpene synthase pair Mv CPS1 and Mv ELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3𝛽-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3𝛽,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum. Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 𝜇g mL⁻¹, while trihydroxydolabrene-mediated inhibition was specific to F. verticillioides. These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.

Background Multiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals. Results The authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed. Conclusions This work demonstrates that different methods using chemical, thermal, and non-destructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Diterpenoids constitute a diverse class of metabolites with critical functions in plant development, defense, and ecological adaptation. Major monocot crops, such as maize (Zea mays) and rice (Oryza sativa), deploy diverse blends of specialized diterpenoids as core components of biotic and abiotic stress resilience. Here, we describe the genome-wide identification and functional characterization of stress-related diterpene synthases (diTPSs) in the dedicated bioenergy crop switchgrass (Panicum virgatum). Mining of the allotetraploid switchgrass genome identified an expansive diTPS family of 31 members, and biochemical analysis of 11 diTPSs revealed a modular metabolic network producing a diverse array of diterpenoid metabolites. In addition to ent-copalyl diphosphate (CPP) and ent-kaurene synthases predictably involved in gibberellin biosynthesis, we identified syn-CPP and ent-labda-13-en-8-ol diphosphate (LPP) synthases as well as two diTPSs forming (+)-labda-8,13E-dienyl diphosphate (8,13-CPP) and ent-neo-cis-trans-clerodienyl diphosphate (CT-CLPP) scaffolds not observed previously in plants. Structure-guided mutagenesis of the (+)-8,13-CPP and ent-neo-CT-CLPP synthases revealed residue substitutions in the active sites that altered product outcome, representing potential neofunctionalization events that occurred during diversification of the switchgrass diTPS family. The conversion of ent-CPP, ent-LPP, syn-CPP, and ent-neo-CT-CLPP by promiscuous diTPSs further yielded distinct labdane-type diterpene olefins and alcohols. Of these metabolites, the formation of 9β-hydroxy-syn-pimar-15-ene and the expression of the corresponding genes were induced in roots and leaves in response to oxidative stress and ultraviolet irradiation, indicating their possible roles in abiotic stress adaptation. Together, these findings expand the known chemical space of diterpenoid metabolism in monocot crops toward systematically investigating and ultimately improving stress resilience traits in crop species.

When a state utility wishes to cross land located within a Native American reservation, but the landowners refuse to allow it, the utility in most circumstances may exercise eminent domain over the land. Under the authority of a federal statute, 25 USC §357, states may generally condemn allotments, plots owned by individuals that lie within the sovereign boundaries of a tribal reservation. Courts have long recognized that the state authority to condemn these allotments under § 357 arises from the principle that individually owned allotments are no longer “tribal” land and, as a result, they are not protected by tribal sovereignty. Congress’s failure to transition away from the allotment system has resulted in an ownership structure for certain plots of reservation land that it did not anticipate when it enacted § 357. Today, not all allotments are held entirely by individuals, and many now contain fractional, undivided interests that belong to tribes themselves. This status of joint ownership between individuals and tribes, which this Comment refers to as “partially tribal,” leads to considerable complications with respect to the scope of § 357. Courts have routinely held that land owned by a sovereign Native American tribe is not subject to state condemnation and that this principle protects tribal interests in allotments. Unresolved, however, is whether a tribal interest in an allotment—which can be as small as a fraction of 1 percent—should immunize even the nontribal interest in the plot from state condemnation proceedings. In other words, should a fractional tribal interest place an entire parcel out of the state’s reach? This Comment argues that it should. The courts that have attempted to allow condemnations to proceed against partially tribal allotments run into the problem that all ownership interests in an allotment are undivided; each owner holds an undivided share of the whole parcel. This means there is no way to divide the tribal interests from the nontribal ones without effecting some kind of incursion on a tribal land interest without the tribe’s or Congress’s consent, a result that principles of tribal sovereignty squarely reject. This Comment recognizes that Congress’s intent when it passed § 357 was to eliminate tribal landholdings, but it argues that Congress has since changed course such that courts should disregard that original intent. This Comment also concedes that diminishing eminent domain power may lead to holdout problems, though it argues that protecting tribal sovereignty is the more important interest. Consistent with the principle that states may not diminish any tribal sovereignty without Congress’s consent, this Comment concludes that a state utility has multiple avenues for seeking access to a partially tribal allotment, including opportunities for negotiation with the tribe and the federal government. Courts should not permit states to use § 357 unilaterally to divest a nonconsenting tribe from its interest in land.

Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.

Lignocellulosic biomass offers a vast and renewable resource for biofuel production and carbon management solutions. The effective conversion of lignocellulosic biomass into economically competitive biofuels and bioproducts demands a comprehensive understanding of its complex structure and composition, often requiring a range of analytical tools to achieve meaningful insights. However, for the analysis of rigid solids, many traditional methods necessitate dissolution or chemical/physical modification of the sample, which limit our ability to capture an intact view of its structural components. This highlights the need for non-destructive approaches, such as solid-state nuclear magnetic resonance (ssNMR), which preserves the sample’s natural state while providing deep, molecular-level insights. While advanced multi-dimensional ssNMR on 13 C-enriched materials has recently proven exceptionally valuable for elucidating the complex macrostructure of biomass, isotopic enrichment is expensive, laborious and is clearly infeasible at large scales. In this review, we explore the role of solid-state NMR methods at natural isotopic abundance as essential tools for the non-destructive, in-depth characterization of lignocellulosic biomass and bioenergy materials in their native and unaltered state. After a brief introduction to the basic principles of solid-state NMR, we first describe the acquisition and interpretation of routine 1D 13 C ssNMR spectra of lignocellulose and other related biopolymers and products. We then delve into more advanced ssNMR approaches, including key spectral editing techniques, probing polymer dynamics, and various 2D methods applicable at natural abundance. Understanding of domain miscibility as observed from proton-based spin diffusion effects is a theme throughout. Our aim is to highlight key examples where ssNMR provides valuable insights into the composition, structure, dynamics, and morphology of rigid biomaterials relevant to the bioenergy economy, revealing both the native structures and fundamental transformations that occur across conversion and decomposition pathways. We hope that this review encourages a broader adoption of ssNMR methods in bioenergy research, where it can serve as a pivotal analytical tool for achieving sustainable biomass utilization and advancing a carbon-efficient bioeconomy.

[This corrects the article DOI: 10.1371/journal.pone.0176507.].[This corrects the article DOI: 10.1371/journal.pone.0176507.].