Biological activities of organic extracts and specialized metabolites from different parts of Cryptomeria japonica (Cupressaceae) - A critical review.

Forest operations and wood industry generate large amounts of residues that are discarded in the field and cause environmental pollution. However, these biomass residues are still raw materials to obtain value-added products, such as essential oils, organic/aqueous extracts and resins that are among the great natural sources of bioactive metabolites. Thus, in recent years, the scientific community is giving special attention to their valorization. To date, different uses of biomass residues have been proposed, such as a source of renewable energy, fertilizers, animal feed and bioactive molecules. In this context, Cryptomeria japonica biomass residues (e.g., bark and its exudate, heartwood, sapwood, leaves, cones and roots) represent a source of diverse specialized metabolites (e.g., sesqui-, di-, tri- and sesquarterpenes, flavonoids, lignans and norlignans) with potential application in different fields, particularly in the agrochemical, food, cosmeceutical, pharmaceutical, phytomedicine and esthetic, due to their valuable multi-bioactivities determined over the last decades. Thus, this review provides an overview of the reported biological activities of organic extracts/fractions and their specialized metabolites obtained from different parts of C. japonica, in order to encourage the alternative uses of C. japonica wastes/byproducts, and implement a sustainable and circular bioeconomy.

Neurotrophic and Immunomodulatory Lanostane Triterpenoids from Wood-Inhabiting Basidiomycota.

Neurotrophins such as nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for the treatment of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors, namely, ngf (sulphurenic acid, 15α-dehydroxytrametenolic acid, fomefficinic acid D, and 16α-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15α-dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as a starting point for the development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may, therefore, be better suited for therapy than many other neurotrophic compounds that were previously reported.

Extracts Prepared from Feed Supplements Containing Wood Lignans Improve Intestinal Health by Strengthening Barrier Integrity and Reducing Inflammation.

Lignans are known to exhibit a broad spectrum of biological activities, indicating their potential as constituents of feed supplements. This study investigated two extracts derived from the feed supplements 'ROI' and 'Protect'-which contain the wood lignans magnolol and honokiol ('ROI'), or soluble tannins additional to the aforementioned lignans ('Protect')-and their impact on selected parameters of intestinal functionality. The antioxidant and anti-inflammatory properties of the extracts were determined by measuring their effects on reactive oxygen species (ROS) and pro-inflammatory cytokine production in vitro. The impact on intestinal barrier integrity was evaluated in Caco-2 cells and Drosophila melanogaster by examining leaky gut formation. Furthermore, a feeding trial using infected piglets was conducted to study the impact on the levels of superoxide dismutase, glutathione and lipid peroxidation. The Protect extract lowered ROS production in Caco-2 cells and reversed the stress-induced weakening of barrier integrity. The ROI extract inhibited the expression or secretion of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα). Moreover, the ROI extract decreased leaky gut formation and mortality rates in Drosophila melanogaster. Dietary supplementation with Protect improved the antioxidant status and barrier integrity of the intestines of infected piglets. In conclusion, wood lignan-enriched feed supplements are valuable tools that support intestinal health by exerting antioxidant, anti-inflammatory and barrier-strengthening effects.

Wood smoke particulate matter (WSPM2.5) induces pyroptosis through both Caspase-1/IL-1ß/IL-18 and ATP/P2Y-dependent mechanisms in human bronchial epithelial cells.

Emerging evidences have linked the air pollution particulate matters, especially the fine particulate matter PM2.5, to the disease development of chronic obstructive pulmonary disease (COPD). Our previous studies reported that biofuel PM2.5 can induce devastated damage of human bronchial epithelial cells, this study aims to further investigate the underlying molecular mechanisms how biofuel PM2.5 induces bronchial epithelial cell death and dysfunction. In this study, biofuel PM2.5 extracted from wood smoke (WSPM2.5) was used according to our previous publication. A 16-HBE cell line was used as the cell model. Results showed that: Firstly, WSPM2.5 induced significant pyroptosis in 16-HBE cells, reflected by the typical changes including elevated release of lactate dehydrogenase release (LDH) and activated activity and expression of Caspase-1/IL-1ß/IL-18 signaling pathway. Then, specific inhibitors for both Caspases (Z-VAD-FMK) and Caspase-1 (VX-765), as well as specific siRNA knockdown of IL-1ß all effectively attenuated the WSPM2.5-induced upregulation of downstream inflammatory cytokines and chemokines (IL-6, IL-8, CXCL-1, CXCL-2, etc), respectively. Notably, WSPM2.5 caused a novel increase of intracellular-to-extracellular ATP secretion, which could also contribute to the WSPM2.5-induced pyroptosis and inflammation by activating the Caspase-1/IL-1ß/IL-18 signaling pathway through possible autocrine and/or paracrine mechanisms. Antagonism of ATP (Apyrase) or specific siRNA knockdown against ATP receptors (P2Y2 and P2Y7) both significantly inhibited the WSPM2.5-induced pyroptosis and inflammation. These results add up to the current knowledge and bring up novel insights that WSPM2.5 could induce significant pyroptosis and inflammation of human bronchial epithelial cells, through both a classic NLRP3/Caspase-1/IL-1ß-dependent and a novel ATP/P2Y-dependent mechanisms.

Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.

Understanding of cell death induced by the constituents of Taxus yunnanensis wood.

The ethanol extract from the wood of Taxus Yunnanensis (TY) induced apoptosis in all cancer cell lines tested, which was mainly due to activation of an extrinsic pathway in human colon cancer DLD-1 cells. The extrinsic pathway was activated by the upregulation of the expression levels of Fas and TRAIL/DR5, which led to the activation of caspase-8. Of note, the machinery of this increase in expression was promoted by the upregulation of MIR32a expression, which silenced MIR34a-targeting E2F3 transcription factor. Furthermore, ectopic expression of MIR32a or siR-E2F3 silencing E2F3 increased Fas and TRAIL/DR5 expression. Thus, the extract activated the extrinsic pathway through the MIR34a/E2F3 axis, resulting in the autocrine and paracrine release of TRAIL, and upregulated expression of death receptors Fas and DR5 in the treated DLD-1 cells, which were functionally validated by Fas immunocytochemistry, and using anti-Fas and anti-TRAIL antibodies, respectively. In vivo, TY showed significant anti-tumor effects on xenografted and syngeneic model mice. The extract may also aid in chemoprevention by selectively making marked tumor cells susceptible to the tumor immunosurveillance system.

Some Minor Characteristics of Spectrophotometric Determination of Antioxidant System and Phenolic Metabolism Enzyme Activity in Wood Plant Tissues of Pinus sylvestris L.

INTRODUCTION: A comprehensive study of enzymes of the antioxidant system (AOS) and phenolic metabolism is an actual subject of biochemical research; changes in the activity of these enzymes can be used as a diagnostic sign. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions. The article presents the chemical kinetics study of reactions catalyzed by superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues (Pinus sylvestris L.). The dependence of the enzyme reaction rate on the enzyme concentration and the substrate concentration is presented, and the pH-optimum for each reaction is established. BACKGROUND: Determination of AOS enzyme activity and PAL activity in woody plants has many difficulties. The chemical composition of pine trunk tissues affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics. OBJECTIVE: This study aimed at determining the AOS enzyme activity in 40-year-old Scots pine (Pinus sylvestris L.) plants growing in the Karelian (Russia) forest seed plantation. METHODS: Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4 °C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 15-min of extraction, the homogenate was centrifuged at 12000 g for 10 min (MPW-351R centrifuge, Poland). The supernatant was purified on 20 cm3 columns with Sephadex G-250. Aliquots with the highest protein amount were collected. In tissues, the protein concentration was 10-50 µg/ml. Proteins in the extracts were quantified by a Bradford assay. The enzyme activity was determined spectrophotometrically on a SpectroStar Nano plate spectrophotometer (BMG Labtech, Germany). RESULTS: Our study made it possible to modify the methods for determining the activity of superoxide dismutase, catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase in Scots pine trunk tissues. The enzymatic reaction rate dependence on the enzyme concentration and the substrate concentration was determined, and pH-optimum was also noted. This methodological article also provides formulas for calculating the activities of the enzymes. CONCLUSION: We found that determining AOS enzyme activity and PAL activity in woody plants is challenging. The chemical composition of the xylem and phloem of pine affects determining AOS enzyme activity and PAL activity. Spectrophotometric determination of AOS enzyme activity and PAL activity gives perfect results when considering all additional controls by taking into account minor characteristics.

Potential use of wood metabolites for cancer treatment.

The study of medicinal plants for cancer treatment has gained attention due to an increasing incidence of cancer worldwide and antineoplastics-related undesirable secondary effects. Most of the natural products of medicinal plants that have been evaluated for cytotoxic activity, are derived from leaves, bark, roots and flowers. However, natural products derived from wood have demonstrated a cytotoxic effect with promising results. Moreover, some fractions and compounds have been isolated of wood in order to increase the effect. This review presents in vitro experimental evidence of cytotoxic effect of natural products from wood against cancer cell lines. It also provides considerations and recommendations to obtain herbal medicines over time.

A Fungal Secretome Adapted for Stress Enabled a Radical Wood Decay Mechanism.

Brown rot fungi release massive amounts of carbon from forest deadwood, particularly at high latitudes. These fungi degrade wood by generating small reactive oxygen species (ROS) to loosen lignocellulose, to then selectively remove carbohydrates. The ROS mechanism has long been considered the key adaptation defining brown rot wood decomposition, but recently, we found preliminary evidence that fungal glycoside hydrolases (GHs) implicated in early cell wall loosening might have been adapted to tolerate ROS stress and to synergize with ROS to loosen woody lignocellulose. In the current study, we found more specifically that side chain hemicellulases that help in the early deconstruction of the lignocellulosic complex are significantly more tolerant of ROS in the brown rot fungus Rhodonia placenta than in a white rot fungus (Trametes versicolor) and a soft rot fungus (Trichoderma reesei). Using proteomics to understand the extent of tolerance, we found that significant oxidation of secreted R. placenta proteins exposed to ROS was less than half of the oxidation observed for T. versicolor or T. reesei. The principal oxidative modifications observed in all cases were monooxidation and dioxidation/trioxidation (mainly in methionine and tryptophan residues), some of which were critical for enzyme activity. At the peptide level, we found that GHs in R. placenta were the least ROS affected among our tested fungi. These results confirm and describe underlying mechanisms of tolerance in early-secreted brown rot fungal hemicellulases. These enzymatic adaptations may have been as important as nonenzymatic ROS pathway adaptations in brown rot fungal evolution. IMPORTANCE Brown rot fungi play a critical role in carbon recycling and are of industrial interest. These fungi typically use reactive oxygen species (ROS) to indiscriminately "loosen" wood cell walls at the outset of decay. Brown rot fungi avoid oxidative stress associated with this ROS step by delaying the expression/secretion of many carbohydrate-active enzymes, but there are exceptions, notably some side chain hemicellulases, implicated in loosening lignocellulose. In this study, we provide enzyme activity and secretomic evidence that these enzymes in the brown rot model Rhodonia placenta are more ROS tolerant than the white and soft rot isolates tested. For R. placenta, and perhaps all brown rot lineages, these ROS tolerance adaptions may have played a long-overshadowed role in enabling brown rot.

Wood transcriptome analysis of Pinus densiflora identifies genes critical for secondary cell wall formation and NAC transcription factors involved in tracheid formation.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

Functionality of Surface Mycelium Interfaces in Wood Bonding.

Filamentous fungi have been considered as candidates to replace petroleum-based adhesives and plastics in novel composite material production, particularly those containing lignocellulosic materials. However, the nature of the role of surface mycelium in the adhesion between lignocellulosic composite components is not well-known. The current study investigated the functionality of surface mycelium for wood bonding by incubating Trametes versicolor on yellow birch veneers and compared the lap-shear strengths after hot-pressing to evaluate if the presence of surface mycelium can improve the interface between two wood layers and consequently improve bonding. We found that the lap-shear strength of the samples was enhanced by the increase of surface mycelium coverage up to 8 days of incubation (up to 1.74 MPa) without a significant wood weight loss. We provide evidence that the bottom surface of the mycelium layer is more hydrophilic, contains more small-scale filamentous structure and contains more functional groups, resulting in better bonding with wood than the top surface. These observations confirm and highlight the functionality of the surface mycelium layer for wood bonding and provide useful information for future developments in fully biobased composites manufacturing.

Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

Testing the elemental defense hypothesis with a woody plant species: Cadmium accumulation protects Populus yunnanensis from leaf herbivory and pathogen infection.

Elemental defense hypothesis states that metals accumulated in plant tissues may serve as defense against herbivores and pathogens. However, evidences collected so far are inconsistent and studies using woody plants as model species are still lacking. In this study we used a local woody plant species, Populus yunnanensis, to investigate whether cadmium (Cd) accumulation in leaves can protect plants from leaf herbivory and pathogen infection. Plants grown with or without Cd supplementation in the soil were subjected to herbivory by a specialist (Botyodes diniasalis) and a generalist (Spodoptera exigua), or to pathogen infection by a leaf pathogenic fungus (Pestalotiopsis microspora). Two additional tests with artificial media amended with a series of Cd concentrations were conducted for S. exigua and P. microspora to investigate the toxicity of Cd independently of other organic defenses present in P. yunnanensis leaves. The results showed that both herbivores strongly preferred control leaves over leaves containing high Cd. Feeding on leaves from Cd-treated plants significantly reduced the growth and survivals of both herbivores. Furthermore, plants grown on Cd-amended soil were more resistant to fungal infection. Growth of S. exigua and P. microspora on artificial media decreased with increasing Cd concentrations. In conclusion, we found that Cd accumulated in P. yunnanensis leaves could effectively reduce leaf herbivory and pathogen infection, which fully supported the Elemental defense hypothesis.

Over-expression of poplar NAC15 gene enhances wood formation in transgenic tobacco.

BACKGROUND: NAC (NAM/ATAF/CUC) is one of the largest plant-specific transcription factor (TF) families known to play significant roles in wood formation. Acting as master gene regulators, a few NAC genes can activate secondary wall biosynthesis during wood formation in woody plants. RESULTS: In the present study, firstly, we screened 110 differentially expressed NAC genes in the leaves, stems, and roots of di-haploid Populus simonii×P. nigra by RNA-Seq. Then we identified a nucleus-targeted gene, NAC15 gene, which was one of the highly expressed genes in the stem among 110 NAC family members. Thirdly, we conducted expression pattern analysis of NAC15 gene, and observed NAC15 gene was most highly expressed in the xylem by RT-qPCR. Moreover, we transferred NAC15 gene into tobacco and obtained 12 transgenic lines overexpressing NAC15 gene (TLs). And the relative higher content of hemicellulose, cellulose and lignin was observed in the TLs compared to the control lines containing empty vector (CLs). It also showed darker staining in the culms of the TLs with phloroglucinol staining, compared to the CLs. Furthermore, the relative expression level of a few lignin- and cellulose-related genes was significantly higher in the TLs than that in the CLs. CONCLUSIONS: The overall results indicated that NAC15 gene is highly expressed in the xylem of poplar and may be a potential candidate gene playing an important role in wood formation in transgenic tobacco.

Genomic content of chemosensory genes correlates with host range in wood-boring beetles (Dendroctonus ponderosae, Agrilus planipennis, and Anoplophora glabripennis).

BACKGROUND: Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species' ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species. RESULTS: We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle (Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP "tetramer", and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the "GR215 clade"), and conservation of IR60a from Drosophila melanogaster. CONCLUSIONS: Our genome annotations represent important quantitative and qualitative improvements of the original datasets derived from transcriptomes of D. ponderosae and A. planipennis, facilitating evolutionary analysis of chemosensory genes in the Coleoptera where only a few genomes were previously annotated. Our analysis suggests a correlation between chemosensory gene content and host specificity in beetles. Future studies should include additional species to consolidate this correlation, and functionally characterize identified proteins as an important step towards improved control of these pests.

Peroxidase evolution in white-rot fungi follows wood lignin evolution in plants.

A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme (k2app and k3app ) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k3app ). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral "tryptophanless" enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units.

Genome Sequences Provide Insights into the Reticulate Origin and Unique Traits of Woody Bamboos.

Polyploidization is a major driver of speciation and its importance to plant evolution has been well recognized. Bamboos comprise one diploid herbaceous and three polyploid woody lineages, and are members of the only major subfamily in grasses that diversified in forests, with the woody members having a tree-like lignified culm. In this study, we generated four draft genome assemblies of major bamboo lineages with three different ploidy levels (diploid, tetraploid, and hexaploid). We also constructed a high-density genetic linkage map for a hexaploid species of bamboo, and used a linkage-map-based strategy for genome assembly and identification of subgenomes in polyploids. Further phylogenomic analyses using a large dataset of syntenic genes with expected copies based on ploidy levels revealed that woody bamboos originated subsequent to the divergence of the herbaceous bamboo lineage, and experienced complex reticulate evolution through three independent allopolyploid events involving four extinct diploid ancestors. A shared but distinct subgenome was identified in all polyploid forms, and the progenitor of this subgenome could have been critical in ancient polyploidizations and the origin of woody bamboos. Important genetic clues to the unique flowering behavior and woody trait in bamboos were also found. Taken together, our study provides significant insights into ancient reticulate evolution at the subgenome level in the absence of extant donor species, and offers a potential model scenario for broad-scale study of angiosperm origination by allopolyploidization.

Stimulation of Wood Degradation by Daedaleopsis confragosa and D. tricolor.

Biological pretreatment of the lignocellulosic residues, in which white-rot fungi have a crucial role, has many advantages compared to the chemical, physical, and physico-chemical methods of delignification and therefore attracts increasing scientific attention. Regarding the fact that properties and capacities of the ligninolytic enzymes of Daedaleopsis spp. are still unknown, the aim of this study was to research how nitrogen sources and inducers affect the potential of Daedaleopsis confragosa and Daedaleopsis tricolor to degrade cherry sawdust. NH4NO3, (NH4)2SO4, and peptone were tested as nitrogen sources, while veratryl alcohol, p-anisidine, vanillic acid, and phenylmethylsulfonyl fluoride were the studied inducers. As Mn-dependent peroxidase and laccase were the leader enzymes and cherry sawdust/peptone medium the best stimulator of their activities, the effect of inducers on delignification potential of these species was studied during fermentation of that substrate. Veratryl alcohol was the best stimulator of laccase and phenylmethylsulfonyl fluoride of Mn-dependent peroxidase activity (27,610.0 and 1338.4 U/L, respectively). These inducers also increased cherry sawdust delignification selectivity, particularly in D. tricolor in the presence of phenylmethylsulfonyl fluoride (lignin:hemicellulose:cellulose = 32.1%:0.9%:11.7%). Owing to the presented results, studied species could have an important role in the phase of lignocellulose pretreatment in various biotechnological processes.

Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals.

Brown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grew Postia placenta directionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H2O2 and Fe2+ concentrations in the extracellular environment were conducive to HO˙ production in early (H2O2:Fe2+ ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producer Trichoderma reesei Collectively, this indicates that P. placenta uses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood.IMPORTANCE Wood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to "loosen" wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H2O2, Fe2+), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

Influence of light on lignin-degrading activities of fungal genus Polyporus s. str.

Six strains belonging to five species of Polyporus (P. arcularius, P. arcularioides, P. tricholoma, P. cfr. tricholoma, and P. varius), collected from an Atlantic Forest area in Misiones (Argentina), where species usually grow exposed to high temperatures and humidity, were identified by morphological and molecular analyses. P. tricholoma (BAFC 4536) and P. arcularioides (BAFC 4534) were selected by their lignin-degrading enzyme production, their ability to produce primordial of basidiomes under submerged fermentation, and the decrease in lignin content caused in Poplar wood (up to 29% after 45 days). Among several variables evaluated with a Plackett-Burman design (glucose, copper, vanillic acid and manganese concentration, incubation period, and light incidence), the most important factor affecting laccase and Mn-peroxidase (MnP) production by both strains, was light incidence. Light induced fruit body development but diminished laccase and MnP production. Moreover, a modified isoenzymatic laccase pattern was observed, showing additional isoenzymes when fungi were cultivated under darkness and differences in optimal temperature. Although the studied strains did not produce high laccase and MnP titers (uppermost detected 4230 and 90 U L-1 , respectively), their laccases showed thermal stability and optimal temperature above 70 °C, representing an interesting source in the search of thermo-tolerant enzymes for biotechnological applications.