Integrated Preparation of Hollow Lignin Nanoparticles as a Drug Carrier and Levulinic Acid from the Poplar Wood Prehydrolysis Liquor.

Prehydrolysis liquid (PHL) from dissolving pulp and biorefinery industries is rich in saccharides and lignin, being considered as a potential source of value-added materials and platform molecules. This study proposed an environmentally friendly and simple method to prepare morphologically controllable hollow lignin nanoparticles (LNPs) and levulinic acid (LA) from PHL. In the first step, after hydrothermal treatment of PHL with p-toluenesulfonic acid (p-TsOH), lignin with a uniform molecular weight was obtained to prepare LNPs. The prepared LNPs have an obvious hollow structure, with an average size of 490-660 nm, and exhibit good stability during 30 days of storage. When the as-obtained LNPs were used as a sustained-release agent for amikacin sulfate, the encapsulation efficiency reached over 70% and the release efficiency within 40 h reached 69.2% in a pH 5.5 buffer. Subsequently, the remaining PHL that contains saccharides was directly used for LA production under the catalysis of p-TsOH. At 150 °C for 1.5 h, the LA yield reached 58.4% and remained at 56% after 5 cycles of p-TsOH. It is worth noting that only p-TsOH was used as a reactive reagent throughout the entire preparation process. Overall, this study provided a novel pathway for the integrated utilization of PHL and showed the immense potential of the preparation and application of LNPs.

Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Lithium and strontium accumulation in native and invasive plants of the Sava River: Implications for bioindication and phytoremediation.

The objective of this study was to investigate the potential of native and invasive plant species for the uptake and accumulation of lithium (Li) and strontium (Sr) along the Sava River, focusing on their bioindication and phytoremediation capabilities. Sampling was carried out in riparian zones exposed to different pollution sources in Slovenia, Croatia, and Serbia. Plant samples of native (Salix alba, Populus alba, Populus nigra, Ulmus glabra, Juglans regia) and invasive (Amorpha fruticosa, Reynoutria japonica, Solidago canadensis, Impatiens glandulifera) species were collected. The content of Li and Sr was analyzed in the soils, roots, and leaves of the selected plants, as well as physical and chemical soil properties. Both Li and Sr content in the soils increased from the source to the mouth of the Sava River. The native species showed significant potential for Li and Sr accumulation based on the metal accumulation index. The highest Sr accumulation was measured in the leaves of Salix alba and the roots of Juglans regia, while the highest Li accumulation was measured in Ulmus glabra. Native species, especially Salix alba, proved to be better bioindicators of Li and Sr. Invasive species, especially Amorpha fruticosa and Impatiens glandulifera, showed a remarkable ability to translocate Sr and Li, respectively, to leaves. These results provide valuable insight into the suitability of plants for biomonitoring soil contamination and potential applications in phytoremediation strategies. In summary, the study shows the importance of native species in the context of the accumulation and bioindication of soil pollution.

Unveiling drastic influence of cross-interactions in hydrothermal carbonization of spirulina with cellulose, lignin or poplar on nature of hydrochar and activated carbon.

Spirulina platensis contains abundant nitrogen-containing organics, which might react with derivatives of cellulose/lignin during hydrothermal carbonization (HTC), probably affecting yield, property of hydrochar, and pore development in activation of hydrochar. This was investigated herein by conducting co-HTC of spirulina platensis with cellulose, lignin, and sawdust at 260 °C and subsequent activation of the resulting hydrochars with K2C2O4 at 800 °C. The results showed that cross-condensation of spirulina platensis-derived proteins with cellulose/lignin-derived ketones and phenolics did take place in the co-HTC, forming more π-conjugated heavier organics, retaining more nitrogen species in hydrochar, reducing yields of hydrochar, making the hydrochar more aromatic and increasing the thermal stability and resistivity towards activation. This enhanced the yield of activated carbon (AC) by 7 %-20 % and significantly increased specific surface area of the AC from activation of hydrochar of spirulina platensis + lignin to 2074.5 m2/g (859.3 m2/g from spirulina platensis only and 1170.1 m2/g from lignin only). Furthermore, more mesopores from activation of hydrochar of spirulina platensis + cellulose (47 %) and more micropores from activation of hydrochar of spirulina + sawdust (93 %) was generated. The AC from spirulina platensis + lignin with the developed pore structures generated sufficient sites for adsorption of tetracycline from aqueous phase and minimized steric hindrance for mass transfer with the abundant mesopores (43 %).

Reductive Conversion Leads to Detoxification of Salicortin-like Chemical Defenses (Salicortinoids) in Lepidopteran Specialist Herbivores (Notodontidae).

Lepidopteran specialist herbivores of the Notodontidae family have adapted to thrive on poplar and willow species (Salicaceae). Previous research showed that Cerura vinula, a member of the Notodontidae family occurring throughout Europe and Asia, uses a unique mechanism to transform salicortinoids, the host plant's defense compounds, into quinic acid-salicylate conjugates. However, how the production of this conjugates relates to the detoxification of salicortinoids and how this transformation proceeds mechanistically have remained unknown. To find the mechanisms, we conducted gut homogenate incubation experiments with C. vinula and re-examined its metabolism by analyzing the constituents of its frass. To estimate the contribution of spontaneous degradation, we examined the chemical stability of salicortinoids and found that salicortinoids were degraded very quickly by midgut homogenates and that spontaneous degradation plays only a marginal role in the metabolism. We learned how salicortinoids are transformed into salicylate after we discovered reductively transformed derivatives, which were revealed to play key roles in the metabolism. Unless they have undergone the process of reduction, salicortinoids produce toxic catechol. We also studied constituents in the frass of the Notodontidae species Cerura erminea, Clostera anachoreta, Furcula furcula, Notodonta ziczac, and Pheosia tremula, and found the same metabolites as those described for C. vinula. We conclude that the process whereby salicortinoids are reductively transformed represents an important adaption of the Notodontidae to their Salicaceae host species.

Environment and Genotype Influence Quantitative and Qualitative Variation in Condensed Tannins in Aspen.

Condensed tannins (CTs) are abundant, ecologically-relevant secondary metabolites in many plants, which respond to variables associated with anthropogenic environmental change. While many studies have reported how genetic and environmental factors affect CT concentrations, few have explored how they influence CT molecular structure. Here, using trembling aspen (Populus tremuloides) as a model organism, we report how foliar CT concentrations, polymer sizes, representation of procyanidins and prodelphinidins, and stereochemistry vary in response to changes in air temperature (warming and freeze damage), air composition (elevated CO2 and O3), soil quality (nutrients and microbiome), and herbivory (mammal and lepidopteran). Use of multiple aspen genotypes enabled assessment of genetic influences on aspen CTs. CT concentration and composition were analyzed by thiolysis-ultra high performance liquid chromatography/mass spectrometry in archived leaf samples from prior experiments. All environmental variables explored except for soil microbiome influenced both CT quantity and quality, with climate factors appearing to have larger effect magnitudes than herbivory. Climate, soil, and herbivory effects varied among genotypes, while air composition effects were consistent across genotypes. Considering that CT properties (concentrations and molecular structures) mediate functions at the organismal through ecosystem scales, intraspecific variation in responses of CT properties to environmental factors could provide a pathway through which environmental change exerts selective pressure on Populus populations. Future studies are needed to identify the molecular-level mechanisms by which environmental factors influence CT concentrations and structures, and to establish their ecological and evolutionary significance.

Deconstruction of biomass enabled by local demixing of cosolvents at cellulose and lignin surfaces.

A particularly promising approach to deconstructing and fractionating lignocellulosic biomass to produce green renewable fuels and high-value chemicals pretreats the biomass with organic solvents in aqueous solution. Here, neutron scattering and molecular-dynamics simulations reveal the temperature-dependent morphological changes in poplar wood biomass during tetrahydrofuran (THF):water pretreatment and provide a mechanism by which the solvent components drive efficient biomass breakdown. Whereas lignin dissociates over a wide temperature range (>25 °C) cellulose disruption occurs only above 150 °C. Neutron scattering with contrast variation provides direct evidence for the formation of THF-rich nanoclusters (Rg ∼ 0.5 nm) on the nonpolar cellulose surfaces and on hydrophobic lignin, and equivalent water-rich nanoclusters on polar cellulose surfaces. The disassembly of the amphiphilic biomass is thus enabled through the local demixing of highly functional cosolvents, THF and water, which preferentially solvate specific biomass surfaces so as to match the local solute polarity. A multiscale description of the efficiency of THF:water pretreatment is provided: matching polarity at the atomic scale prevents lignin aggregation and disrupts cellulose, leading to improvements in deconstruction at the macroscopic scale.

Chemical Composition, Antioxidant Activity, and Antibacterial Activity of Black Poplar Buds' Hydroalcoholic Macerates from Dobrogea Area.

Black poplar buds have high contents of many compounds with therapeutic potential, which are useful in cosmetics and the treatment of various dermatitis, respiratory diseases, etc. The aim of this study was to identify and exploit the local plant resources with biologically active properties from the Dobrogea area, Romania. For this purpose, materials were collected from the mentioned area, and macerates of black poplar were prepared in order to evaluate their qualities as antioxidant and antimicrobial agents. Three different black poplar buds' hydroalcoholic macerates were analyzed by the Folin-Ciocâlteau method to estimate the total content of phenolic compounds, by the HPLC-DAD method for identification and quantification of the main bioactive compounds and by the DPPH radical scavenging method to evaluate the antioxidant activity. All hydroalcoholic macerates showed high concentrations of phenolic compounds, the main individual compounds being gallic acid, chlorogenic acid, cinnamic acid, and methyl gallic acid. The antioxidant activity of the black poplar buds' hydroalcoholic macerates, evaluated by the DPPH radical scavenging test, showed high values, between 496 and 1200 mg GAE /100 g d.w. The Cd, Cu, Zn, Ni, and Pb concentrations released in dry poplar buds, determined by AAS, were below the detection limits. Hydroalcoholic macerates of black poplar were tested against two groups of gram-positive bacteria (Enterococcus and Staphylococcus) using an agar well diffusion assay. The in vitro inhibitory activities of the macerates were important and ranged from 8.2-9.4 mm inhibition zones (Staphylococcus) to 8.6 -10 mm inhibition zones (Enterococcus).

p-Coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification.

The enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins. Here, we analyzed transgenic poplars (Populus tremula × Populus alba) expressing the Brachypodium (Brachypodium distachyon) p-coumaroyl-Coenzyme A monolignol transferase 1 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (pCA). Several BdPMT1-OE/WT and BdPMT1-OE/AtF5H-OE lines were grown in the greenhouse, and BdPMT1 expression in xylem was confirmed by RT-PCR. Analyses of poplar stem cell walls (CWs) and of the corresponding purified dioxan lignins (DLs) revealed that BdPMT1-OE lignins were as p-coumaroylated as lignins from C3 grass straws. For some transformants, pCA levels reached 11 mg·g-1 CW and 66 mg·g-1 DL, exceeding levels in Brachypodium or wheat (Triticum aestivum) samples. This unprecedentedly high lignin p-coumaroylation affected neither poplar growth nor stem lignin content. Interestingly, p-coumaroylation of poplar lignins was not favored in BdPMT1-OE/AtF5H-OE transgenic lines despite their high frequency of syringyl units. However, lignins of all BdPMT1-OE lines were structurally modified, with an increase of terminal unit with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples improved the saccharification yield of alkali-pretreated CW, demonstrating that the genetically driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to alkaline treatments used during the industrial processing of lignocellulosics.

A peroxisomal ß-oxidative pathway contributes to the formation of C6-C1 aromatic volatiles in poplar.

Benzenoids (C6-C1 aromatic compounds) play important roles in plant defense and are often produced upon herbivory. Black cottonwood (Populus trichocarpa) produces a variety of volatile and nonvolatile benzenoids involved in various defense responses. However, their biosynthesis in poplar is mainly unresolved. We showed feeding of the poplar leaf beetle (Chrysomela populi) on P. trichocarpa leaves led to increased emission of the benzenoid volatiles benzaldehyde, benzylalcohol, and benzyl benzoate. The accumulation of salicinoids, a group of nonvolatile phenolic defense glycosides composed in part of benzenoid units, was hardly affected by beetle herbivory. In planta labeling experiments revealed that volatile and nonvolatile poplar benzenoids are produced from cinnamic acid (C6-C3). The biosynthesis of C6-C1 aromatic compounds from cinnamic acid has been described in petunia (Petunia hybrida) flowers where the pathway includes a peroxisomal-localized chain shortening sequence, involving cinnamate-CoA ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD), and 3-ketoacyl-CoA thiolase (KAT). Sequence and phylogenetic analysis enabled the identification of small CNL, CHD, and KAT gene families in P. trichocarpa. Heterologous expression of the candidate genes in Escherichia coli and characterization of purified proteins in vitro revealed enzymatic activities similar to those described in petunia flowers. RNA interference-mediated knockdown of the CNL subfamily in gray poplar (Populus x canescens) resulted in decreased emission of C6-C1 aromatic volatiles upon herbivory, while constitutively accumulating salicinoids were not affected. This indicates the peroxisomal ß-oxidative pathway participates in the formation of volatile benzenoids. The chain shortening steps for salicinoids, however, likely employ an alternative pathway.

Evaluation of VOCs Emitted from Biomass Combustion in a Small CHP Plant: Difference between Dry and Wet Poplar Woodchips.

The combustion of biomass is a process that is increasingly used for the generation of heat and energy through different types of wood and agricultural waste. The emissions generated by the combustion of biomass include different kinds of macro- and micropollutants whose formation and concentration varies according to the physical and chemical characteristics of the biomass, the combustion conditions, the plants, and the operational parameters of the process. The aim of this work is to evaluate the effect of biomass moisture content on the formation of volatile organic compounds (VOCs) during the combustion process. Wet and dry poplar chips, with a moisture content of 43.30% and 15.00%, respectively, were used in a cogeneration plant based on a mobile grate furnace. Stack's emissions were sampled through adsorbent tubes and subsequently analyzed by thermal desorption coupled with the GC/MS. The data obtained showed that, depending on the moisture content of the starting matrix, which inevitably influences the quality of combustion, there is significant variation in the production of VOCs.

Phytochemical Profile, Plant Precursors and Some Properties of Georgian Propolis.

Propolis (bee glue) is a resinous substance produced by different species of bees i.a. from available plant resins, balsams, and exudates. It is characterized by significant biological activity (e.g., antimicrobial and antioxidant) and phytochemical diversity related to the available plant sources in specific geographical regions. The available scientific literature on propolis is quite extensive; however, there are only a few reports about propolis originating from Georgia. Therefore, our research was focused on the characterization of Georgian propolis in terms of phytochemical composition and antimicrobial/antioxidant activity. Performed research included UHPLC-DAD-MS/MS phytochemical profiling, determination of total phenolic and flavonoid content, antiradical and antioxidant activity (DPPH and FRAP assays) as well as antibacterial activity of propolis extracts obtained using 70% ethanol (70EE). Georgian propolis extracts exhibited strong activity against Gram-positive bacteria (22 mm-disc assay/64 µg/mL-MIC for S. aureus, sample from Imereti) and weaker against Gram-negative strains as well as strong antioxidant properties (up to 117.71 ± 1.04 mgGAE/g in DPPH assay, up to 16.83 ± 1.02 mmol Fe2+/g in FRAP assay for samples from Orgora and Qvakhreli, respectively). The phytochemical profile of Georgian propolis was characterized by the presence of flavonoids, free phenolic acids, and their esters. In most of the samples, flavonoids were the main chemical group (52 compounds), represented mainly by 3-O-pinobanksin acetate, pinocembrin, chrysin, galangin, and pinobanksin. The primary plant precursor of the Georgian bee glue is black poplar (Populus nigra L.) while the secondary is aspen poplar (P. tremula L.).

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Oxidative Catalytic Fractionation of Lignocellulosic Biomass under Non-alkaline Conditions.

Biomass pretreatment methods are commonly used to isolate carbohydrates from biomass, but they often lead to modification, degradation, and/or low yields of lignin. Catalytic fractionation approaches provide a possible solution to these challenges by separating the polymeric sugar and lignin fractions in the presence of a catalyst that promotes cleavage of the lignin into aromatic monomers. Here, we demonstrate an oxidative fractionation method conducted in the presence of a heterogeneous non-precious-metal Co-N-C catalyst and O2 in acetone as the solvent. The process affords a 15 wt% yield of phenolic products bearing aldehydes (vanillin, syringaldehyde) and carboxylic acids (p-hydroxybenzoic acid, vanillic acid, syringic acid), complementing the alkylated phenols obtained from existing reductive catalytic fractionation methods. The oxygenated aromatics derived from this process have appealing features for use in polymer synthesis and/or biological funneling to value-added products, and the non-alkaline conditions associated with this process support preservation of the cellulose, which remains insoluble at reaction conditions and is recovered as a solid.

Adaptive variation and plasticity in non-structural carbohydrate storage in a temperate tree species.

Trees' total amount of non-structural carbohydrate (NSC) stores and the proportion of these stores residing as insoluble starch are vital traits for individuals living in variable environments. However, our understanding of how stores vary in response to environmental stress is poorly understood as the genetic component of storage is rarely accounted for in studies. Here, we quantified variation in NSC traits in branch samples taken from over 600 clonally transplanted black cottonwood (Populus trichocarpa) trees grown in two common gardens. We found heritable variation in both total NSC stores and the proportion of stores in starch (H2TNC  = 0.19, H2PropStarch  = 0.31), indicating a substantial genetic component of variation. In addition, we found high amounts of plasticity in both traits in response to cold temperatures and significant genotype-by-environment (GxE) interactions in the total amount of NSC stored (54% of P is GxE). This finding of high GxE indicates extensive variation across trees in their response to environment, which may explain why previous studies of carbohydrate stores' responses to stress have failed to converge on a consistent pattern. Overall, we found high amounts of environmental and genetic variation in NSC storage concentrations, which may bolster species against future climate change.

Root Secondary Metabolites in Populus tremuloides: Effects of Simulated Climate Warming, Defoliation, and Genotype.

Climate warming can influence interactions between plants and associated organisms by altering levels of plant secondary metabolites. In contrast to studies of elevated temperature on aboveground phytochemistry, the consequences of warming on root chemistry have received little attention. Herein, we investigated the effects of elevated temperature, defoliation, and genotype on root biomass and phenolic compounds in trembling aspen (Populus tremuloides). We grew saplings of three aspen genotypes under ambient or elevated temperatures (+4-6 °C), and defoliated (by 75%) half of the trees in each treatment. After 4 months, we harvested roots and determined their condensed tannin and salicinoid (phenolic glycoside) concentrations. Defoliation reduced root biomass, with a slightly larger impact under elevated, relative to ambient, temperature. Elevated temperature decreased condensed tannin concentrations by 21-43% across the various treatment combinations. Warming alone did not alter salicinoid concentrations but eliminated a small negative impact of defoliation on those compounds. Graphical vector analysis suggests that effects of warming and defoliation on condensed tannins and salicinoids were predominantly due to reduced biosynthesis of these metabolites in roots, rather than to changes in root biomass. In general, genotypes did not differ in their responses to temperature or temperature by defoliation interactions. Collectively, our results suggest that future climate warming will alter root phytochemistry, and that effects will vary among different classes of secondary metabolites and be influenced by concurrent ecological interactions such as herbivory. Temperature- and herbivory-mediated changes in root chemistry have the potential to influence belowground trophic interactions and soil nutrient dynamics.

Antioxidant Activity and Chemical Characteristics of Supercritical CO2 and Water Extracts from Willow and Poplar.

Renewable feedstock from perennial industrial crops, including those cultivated on marginal land in a short-rotation coppice system, could be an important contribution to the bioeconomy. The majority of data available on the topic are limited to the production of bioenergy from this type of biomass. According to the concept of bioeconomy, biomass-based bioproducts have priority over energy production. This paper characterizes the chemical composition and antioxidant activity of extracts from bark (b), wood (w) or a mixture of bark and wood (b + w) from Salix purpurea, Salix viminalis and Populus nigra obtained using supercritical carbon dioxide (scCO2), scCO2 and water (1%, w/w) or only water. Generally, a high concentration of polyphenols was obtained after extraction with scCO2 and water, while the lowest concentration was found in extracts obtained with scCO2. The highest concentration of polyphenols (p < 0.05) was obtained in an extract from P. nigra (b) (502.62 ± 9.86 mg GAE/g dry matter (d.m.)) after extraction with scCO2 and water, whereas the lowest polyphenol concentration was observed in an scCO2 extract from S. purpurea (b) (6.02 ± 0.13 mg GAE/g d.m.). The flavonoids were effectively separated by extraction with scCO2 (0.88-18.37 mg QE/g d.m.). A positive linear relationship between the antioxidant activity determined by DPPH and ABTS assays and the concentration of polyphenols was demonstrated, R2 = 0.8377 and R2 = 0.9568, respectively. It is most probable that the concentration of flavonoids, rather than the concentration of polyphenols, determines the chelating activity of Fe2+. The Fe2+-chelating activity of scCO2 extracts ranged from 75.11% (EC50 = 5.41 mg/cm3, S. purpurea, b + w) to 99.43% (EC50 = 0.85 mg/cm3, P. nigra, b + w). The lowest chelating activity was demonstrated by the extracts obtained with scCO2 and water (maximum 26.36%, S. purpurea, b + w). In extracts obtained with scCO2 and water, p-hydroxybenzoic acid (210-428 µg/g), p-coumaric acid (56-281 µg/g), saligenin (142-300 µg/g) and salicortin (16-164 µg/g) were the dominant polyphenols. All of these chemical compounds occurred mainly in the free form. The S. purpurea, S. viminalis and P. nigra biomass proved to be an attractive source of biologically active compounds for various possible applications in food, drugs or cosmetics. These compounds could be extracted using an environmentally friendly method with scCO2 and water as a co-solvent.

A Viable New Strategy for the Discovery of Peptide Proteolytic Cleavage Products in Plant-Microbe Interactions.

Small peptides that are proteolytic cleavage products (PCPs) of less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry-based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy, optimized specifically for small PCPs, using state-of-the-art de novo-assisted peptide sequencing. As a case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus × canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor. In total, we identified 1,660 and 2,870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the photosystem II complex 5-kDa protein (approximately 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared with root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor. These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.

Mild Acetylation and Solubilization of Ground Whole Plant Cell Walls in EmimAc: A Method for Solution-State NMR in DMSO-d6.

Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing the structure of lignocellulose, which involve sequential extraction and characterization of the resulting fractions, are time-consuming and labor-intensive. The present study describes a new and facile system for rationally derivatizing and dissolving coarsely ground plant cell wall materials. Using ionic liquids (EmimAc) and dichloroacetyl chloride as a solvent/reagent produced mildly acetylated whole cell walls without significant degradation. The acetylated products were soluble in DMSO-d6 from which they can be characterized by solution-state two-dimensional nuclear magnetic resonance (2D NMR) spectrometry. A distinct advantage of the procedure is that it realizes the dissolution of whole lignocellulosic materials without requiring harsh ball milling, thereby allowing the acquisition of high-resolution 2D NMR spectra to revealing structural details of the main components (lignin and polysaccharides). The method is therefore beneficial to understanding the composition and structure of biomass aimed at its improved utilization.

Isotopic composition and concentration of total nitrogen and nitrate in xylem sap under near steady-state hydroponics.

After root uptake, nitrate is effluxed back to the medium, assimilated locally, or translocated to shoots. Rooted black cottonwood (Populus trichocarpa) scions were supplied with a NO3- -based (0.5 mM) nutrient medium of known isotopic composition (δ15 N), and xylem sap was collected by pressure bombing. To establish a sampling protocol, sap was collected from lower and upper stem sections at 0.1-0.2 MPa above the balancing pressure, and after increasing the pressure by a further 0.5 MPa. Xylem sap from upper stem sections was partially diluted at higher pressure. Further analysis was restricted to sap obtained from intact shoots at low pressure. Total-, NO3- -N and, by difference, organic-N concentrations ranged from 6.1-11.0, 1.2-2.4, and 4.6-9.4 mM, while discrimination relative to the nutrient medium was -6.3 to 0.5‰, -23.3 to -11.5‰ and - 1.3 to 4.9‰, respectively. There was diurnal variation in δ15 N of total- and organic-N, but not NO3- . The difference in δ15 N between xylem NO3- and organic-N suggests that discrimination by nitrate reductase is near 25.1 ± 1.6‰. When this value was used in an isotope mass balance model, the predicted xylem sap NO3- -N to total-N ratio closely matched direct measurement.