Cell Wall Polymers Research Articles

Cell wall biology of the moss Physcomitrium patens.

The moss Physcomitrium (previously Physcomitrella) patens is a non-vascular plant belonging to the bryophytes that has been used as a model species to study the evolution of plant cell wall structure and biosynthesis. Here, we present an updated review of the cell wall biology of P. patens. Immunocytochemical and structural studies have shown that the cell walls of P. patens mainly contain cellulose, hemicelluloses (xyloglucan, xylan, glucomannan, and arabinoglucan), pectin, and glycoproteins, and their abundance varies among different cell types and at different plant developmental stages. Genetic and biochemical analyses have revealed that a number of genes involved in cell wall biosynthesis are functionally conserved between P. patens and vascular plants, indicating that the common ancestor of mosses and vascular plants had already acquired most of the biosynthetic machinery to make various cell wall polymers. Although P. patens does not synthesize lignin, homologs of the phenylpropanoid biosynthetic pathway genes exist in P. patens and they play an essential role in the production of caffeate derivatives for cuticle formation. Further genetic and biochemical dissection of cell wall biosynthetic genes in P. patens promises to provide additional insights into the evolutionary history of plant cell wall structure and biosynthesis.

New facts about callose events in the young ovules of some sexual and apomictic species of the Asteraceae family.

Callose (β-1,3-glucan) is one of the cell wall polymers that plays an important role in many biological processes in plants, including reproductive development. In angiosperms, timely deposition and degradation of callose during sporogenesis accompanies the transition of cells from somatic to generative identity. However, knowledge on the regulation of callose biosynthesis at specific sites of the megasporocyte wall remains limited and the data on its distribution are not conclusive. Establishing the callose deposition pattern in a large number of species can contribute to full understanding of its function in reproductive development. Previous studies focused on callose events in sexual species and only a few concerned apomicts. The main goal of our research was to establish and compare the pattern of callose deposition during early sexual and diplosporous processes in the ovules of some Hieracium, Pilosella and Taraxacum (Asteraceae) species; aniline blue staining technique was used for this purpose. Our findings indicate that callose deposition accompanies both meiotic and diplosporous development of the megaspore mother cell. This suggests that it has similar regulatory functions in intercellular communication regardless of the mode of reproduction. Interestingly, callose deposition followed a different pattern in the studied sexual and diplosporous species compared to most angiosperms as it usually began at the micropylar pole of the megasporocyte. Here, it was only in sexually reproducing H. transylvanicum that callose first appeared at the chalazal pole of the megasporocyte. The present paper additionally discusses the occurrence of aposporous initial cells with callose-rich walls in the ovules of diploid species.

Extracellular vesiculo-tubular structures associated with suberin deposition in plant cell walls

Suberin is a fundamental plant biopolymer, found in protective tissues, such as seed coats, exodermis and endodermis of roots. Suberin is deposited in most suberizing cells in the form of lamellae just outside of the plasma membrane, below the primary cell wall. How monomeric suberin precursors, thought to be synthesized at the endoplasmic reticulum, are transported outside of the cell, for polymerization into suberin lamellae has remained obscure. Using electron-microscopy, we observed large numbers of extracellular vesiculo-tubular structures (EVs) to accumulate specifically in suberizing cells, in both chemically and cryo-fixed samples. EV presence correlates perfectly with root suberization and we could block suberin deposition and vesicle accumulation by affecting early, as well as late steps in the secretory pathway. Whereas many previous reports have described EVs in the context of biotic interactions, our results suggest a developmental role for extracellular vesicles in the formation of a major cell wall polymer.

The spatial orientation and interaction of cell wall polymers in bamboo revealed with a combination of imaging polarized FTIR and directional chemical removal

The mechanical and physical properties of lignocellulosic materials are closely related to the orientation and interaction of the polymers within cell walls. In this work, Imaging Polarized FTIR, combined with directional chemical removal, was applied to characterize the spatial orientation and interaction of cell wall polymers in bamboo fibers and parenchyma cells from two bamboo species. The results demonstrate the cellulose in bamboo fibers is nearly axially oriented whereas it is almost transversely arranged in parenchyma cells. Xylan and lignin are both preferentially oriented alongside cellulose, but with less orientation degree in the parenchyma cells. After lignin removal, the average orientation of xylan and cellulose is little affected, suggesting a strong interaction between cellulose and xylan. However, the alkaline treatment significantly weakens the orientation of lignin in both fibers and parenchyma cells, and more significant for the latter, indicating the easy-degradable nature of lignin in parenchyma cells. Additionally, it seemed the lignin and xylan in fibers were more difficult to remove as compared to parenchyma cells, supporting the assumption that stronger interaction exists between lignin and xylan in the fibers. In a word, it is believed parenchyma cells are more suitable for biorefinery owing to their less ordered and relatively loose molecular assembly, as compared to fibers.Graphical abstract

Toward bioeconomy of a multipurpose cereal: Cell wall chemistry of Sorghum is largely buffered against stem sugar content

AbstractBackground and ObjectivesSorghum (Sorghum bicolor L. Moench) is a multipurpose crop with high potential for the bioeconomy. Ten sweet, grain and dual‐purpose sorghum genotypes were compared with respect to sugar‐related traits and cell wall composition.FindingsThe dual‐purpose hybrids Ganymed, Zerberus, and Tarzan performed better with respect to plant height, cane weight, and juice volume, but the sweet genotype KIT1 produced the highest sugar concentration (°Brix) in the stem. Analytical sugar data demonstrate genotypic differences in terms of sugar composition and concentrations, with sucrose being dominant. The monosaccharide composition of the stem cell wall polysaccharides showed surprisingly little genotypic variability. Variations in contents of lignin and cell wall‐bound hydroxycinnamate monomers were moderate.ConclusionsSweet, grain and dual‐purpose sorghum genotypes differ widely in terms of sugar‐related morphological parameters but are comparable with respect to their cell wall chemistry.Significance and NoveltyThe use of sorghum as a bioeconomy crop has mainly focused on the extraction of sugar for bioethanol production. However, besides cell wall polymers, the potential usage of hydroxycinnamates as platform molecules for the chemical industry may improve the valorization of the residues after sugar extraction. This application appears to be fairly independent of genotype, further increasing the potential of sorghum for the bioeconomy.

Dynamic imaging of cell wall polysaccharides by metabolic click-mediated labeling of pectins in living elongating cells.

Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Detection and Analysis of Syntenic Quantitative Trait Loci Controlling Cell Wall Quality in Angiosperms.

Translational genomics can enable a quicker improvement of orphan crops toward novel agricultural applications, including the advancement of orphan biomass species for cultivation on marginal lands. In this sense, cell wall quality is a preeminent breeding target. However, tools to efficiently project genetic data on target traits across large sets of species are currently missing. This study aimed at closing this gap by developing a strategy to project a set of cell wall QTLs across a large group of plants by using genome synteny. This strategy is suited for large-scale analyses and detected 362 syntenic cell wall QTLs (SQTLs) across 74 angiosperms, including several (orphan) biomass species. SQTLs analyses revealed that they span large portions of the initial cell wall QTLs and are extensively conserved across diverse species. Moreover, numerous QTLs cell wall genes were conserved through SQTLs, including genes displaying allelic variation associated with cell wall composition. Functional analyses showed that highly conserved genes of SQTLs include important cell wall transcription factors and genes involved in the remodeling of cell wall polymers. For some of these gene families, SQTLs indicated the presence of differentially conserved genomic contexts for different gene members, highlighting their utility as a tool to pinpoint gene targets that maximize the likelihood of functional gene conservation. Overall, the results of this study can facilitate “universal” approaches for breeding (orphan) biomass crops, while the strategy for QTLs translation can be applied to other sets of traits and species, helping to unlock the potential of orphan species.

FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits. Transcriptomic analysis indicated that the expression of pectate lyase A (FvPLA) was significantly downregulated in the FvWRKY48-RNAi receptacle. We determined that FvWRKY48 bound to the FvPLA promoter via a W-box element through yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation quantitative polymerase chain reaction experiments, and β-glucosidase activity assays suggested that this binding promotes pectate lyase activity. In addition, softening and pectin degradation were more intense in FvPLA-OE fruit than in EV fruit, and the middle lamella and tricellular junction zones were denser in FvPLA-RNAi fruit than in EV fruit. We speculated that FvWRKY48 maybe increase the expression of FvPLA, resulting in pectin degradation and fruit softening.

Graphene as reinforcing filler in polyvinyl acetate resin

Graphene gel and powder were added to polyvinyl acetate (PVA) resin to investigate its potential improving effects on shear strength of the resin. Graphene flakes were synthesized based on a simultaneous electrochemical exfoliation/deposition procedure. Flakes were decorated in face with magnetic nanocrystals in hetero-structure. They were mixed with PVA resin at 3% content, based on the wet weight of the resin. Beech wood specimens (Fagus orientalis L.) were prepared according to ASTM D143-14. Separate sets of specimens were prepared to be first heated at 165 °C and 185 °C for 4 h. Results demonstrated significant improving effect of graphene powder on shear strength of PVA resin. Graphene gel did not improve shear strength due to dilution of resin. Density functional theory simulations indicated very low adsorption energy of either pure graphene (−0.45 eV) or Ni-doped graphene (−0.91 eV) flakes on cellulose. Therefore, the improving effect was not attributed to formation of new bonds between graphene flakes and cell-wall polymers. It was concluded that graphene powder acted as a reinforcing filler in the PVA resin matrix, significantly improving its shear strength in the un-heated beech specimens. However, as a result of thermal degradation of cell-wall polymers and the consequent decrease in mechanical properties of the wood, as well as a considerable decrease in wood wettability, failures occurred within the wood structure of the heat-treated specimens rather than within the bond line. Therefore, graphene powder could not improve the overall shear strength in the heat-treated specimens at 185 °C.

High molecular weight chitosan oligosaccharide exhibited antifungal activity by misleading cell wall organization via targeting PHR transglucosidases

The fungal cell wall is an ideal target for the design of antifungal drugs. In this study we used an analog of cell wall polymer, a highly deacetylated high molecular-weight chitosan oligosaccharide (HCOS), to test its effect against pathogenic Candida strains. Results showed that HCOS was successfully incorporated into the dynamic cell wall organization process and exhibited an apparent antifungal activity against both plankton and mature fungal biofilm, by impairing the cell wall integrity. Unexpectedly, mechanistic studies suggested that HCOS exerts its activity by interfering with family members of PHR β-(1,3)-glucanosyl transferases and affecting the connection and assembly of cell wall polysaccharides. Furthermore, HCOS showed great synergistic activity with different fungicides against Candida cells, especially those in biofilm. These findings indicated HCOS has a great potential as an antifungal drug or drug synergist and proposed a novel antifungal strategy with structure-specific oligosaccharides mimicking cell wall polysaccharide fragments.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Heat treatment induces chemical changes and silica sol penetration in wood for properties improvement: hydrophobicity, thermal stability, and surface hardness

Thermal modification could act as pretreatment for promoting silica sol impregnation in wood. It could create extra available channels and spaces for silica transportation and deposition owing to the presence of micro-cracks and enlarged pore size. The significant improvements on anti-hygroscopicity, hydrophobicity, and dimensional stability of treated wood were attributed to the degradation of cell wall polymer (hemicellulose, cellulose, and lignin) and filling effect of silica in wood. The synergistic effect contained the reduction on –OH concentration owing to thermal degradation, and consumption of –OH groups via the interaction (Si–O–Si cross linked networks and Si–O–C covalent bonds) between silica and wood matrix. Furthermore, the degradation of cell wall polymers resulted in the increase of crystallinity in wood. However, the crystallinity turned to decrease due to the amorphous silica impregnation. The silica impregnation could compensate the mass loss caused by thermal degradation, leading to various weight percentage gains (WPGs) that depended on treatment temperature. Moreover, owing to the coverage and penetration of mineral silica in wood, acting as a thermal barrier, the thermal stability and surface hardness of treated wood improved prominently.

The moisture plasticizing effect on enzyme-catalyzed reactions in model and real systems in view of legume ageing and their hard to cook development

This study aims to understand the role of storage conditions (temperature, T and moisture content) in the reactions involved in the cation-pectin-phytate hypothesis, postulated to cause the Hard-to-cook (HTC) defect in beans as influenced by the plasticizing effect of moisture (glass transition ( T g ) concept). The enzyme catalysed reactions, demethylesterification of pectin and hydrolysis of phytic acid, were monitored respectively in simplified pectin and phytic acid/protein model systems. In addition, both reactions were studied in a real system, red kidney bean cotyledon during storage at 35 °C and different relative humidity conditions (6%, 54%, 66%, 72% and 82%) for 15 weeks. The T g -moisture relation of the model systems and the bean cotyledon were established by Dynamic Mechanical Thermal Analysis (DMTA). The T g -line, fitted using the Gordon-Taylor equation, was combined with the moisture sorption isotherm to create a stability diagram. The stability diagram obtained can be used to ascertain the biochemical stability during post-harvest ageing of beans. Results displayed that there was significant hydrolysis of phytic acid in the phytic acid/protein model system and the real system when stored above their glass transition values. On the other hand, in the model system the pectin degree of methylesterifcation (DM) did not significantly change during storage. In the real system, significant, although small, changes of the DM were noted when T - T g > 20 °C. An examination of the glass transition lines revealed that the reactions were influenced by the plasticizing effect of moisture. • The reactions in the cation-pectin-phytate hypothesis were studied in model and real systems. • Hydrolysis of phytic acid is controlled by glass transition. • Glass transition lines of red kidney bean cotyledon and its' cell wall polymers were created. • DMTA based T g lines are better suited for explaining (bio)chemical stability rather than TMCT. • Stability map supports in establishing suitable post-harvest storage conditions.

Biochemical and Histological Insights into the Interaction Between the Canker Pathogen Neofusicoccum parvum and Prunus dulcis.

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars ('Butte', 'Carmel', 'Monterey', and 'Nonpareil'), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was triggered only by fungal infection, not by wounding. A two-dimensional nuclear magnetic resonance and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared with healthy, noninfected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum colonized mainly the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the compartmentalization of decay in trees model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.

Modern Methods of Pre-Treatment of Plant Material for the Extraction of Bioactive Compounds.

In this review, recent advances in the methods of pre-treatment of plant material for the extraction of secondary metabolites with high biological activity are presented. The correct preparation of the material for extraction is as important as the selection of the extraction method. This step should prevent the degradation of bioactive compounds as well as the development of fungi and bacteria. Currently, the methods of preparation are expected to modify the particles of the plant material in such a way that will contribute to the release of bioactive compounds loosely bonded to cell wall polymers. This review presents a wide range of methods of preparing plant material, including drying, freeze-drying, convection drying, microwave vacuum drying, enzymatic processes, and fermentation. The influence of the particular methods on the structure of plant material particles, the level of preserved bioactive compounds, and the possibility of their release during the extraction were highlighted. The plant material pre-treatment techniques used were discussed with respect to the amount of compounds released during extraction as well their application in various industries interested in products with a high content of biologically active compounds, such as the pharmaceutical, cosmetics, and food industries.

Solid-State Nuclear Magnetic Resonance as a Tool to Probe the Impact of Mechanical Preprocessing on the Structure and Arrangement of Plant Cell Wall Polymers.

Efficient separation of the plant cell wall polymers during lignocellulose processing has been historically challenging due to insolubility of the polymers and their propensity for recalcitrant reassembly. Methods, such as “lignin first” extraction techniques, have advanced efficient biomass use, but the molecular mechanisms for recalcitrance remain enigmatic. Here, we discuss how solid-state Nuclear Magnetic Resonance (NMR) approaches report on the 3D organization of cellulose, xylan, and lignin in the plant cell wall. Recent results illustrate that the organization of these polymers varies across biomass sources and sample preparation methods, with even minimal physical processing causing significant effects. These structural differences contribute to variable extraction efficiencies for bioproducts after downstream processing. We propose that solid-state NMR methods can be applied to follow biomass processing, providing an understanding of the polymer rearrangements that can lead to poor yields for the desired bioproducts. The utility of the technique is illustrated for mechanical processing using lab-scale vibratory ball milling of Sorghum bicolor.

The essential role of fungal peroxisomes in plant infection.

Several filamentous fungi are ecologically and economically important plant pathogens that infect a broad variety of crops. They cause high annual yield losses and contaminate seeds and fruits with mycotoxins. Not only powerful infection structures and detrimental toxins, but also cell organelles, such as peroxisomes, play important roles in plant infection. In this review, we summarize recent research results that revealed novel peroxisomal functions of filamentous fungi and highlight the importance of peroxisomes for infection of host plants. Central for fungal virulence are two primary metabolic pathways, fatty acid β‐oxidation and the glyoxylate cycle, both of which are required to produce energy, acetyl‐CoA, and carbohydrates. These are ultimately needed for the synthesis of cell wall polymers and for turgor generation in infection structures. Most novel results stem from different routes of secondary metabolism and demonstrate that peroxisomes produce important precursors and house various enzymes needed for toxin production and melanization of appressoria. All these peroxisomal functions in fungal virulence might represent elegant targets for improved crop protection.

Nested Formation of Calcium Carbonate Polymorphs in a Bacterial Surface Membrane with a Graded Nanoconfinement: An Evolutionary Strategy to Ensure Bacterial Survival.

It is the intention of this study to elucidate the nested formation of calcium carbonate polymorphs or polyamorphs in the different nanosized compartments. With these observations, it can be concluded how the bacteria can survive in a harsh environment with high calcium carbonate supersaturation. The mechanisms of calcium carbonate precipitation at the surface membrane and at the underlying cell wall membrane of the thermophilic soil bacterium Geobacillus stearothermophilus DSM 13240 have been revealed by high-resolution transmission electron microscopy and atomic force microscopy. In this Gram-positive bacterium, nanopores in the surface layer (S-layer) and in the supporting cell wall polymers are nucleation sites for metastable calcium carbonate polymorphs and polyamorphs. In order to observe the different metastable forms, various reaction times and a low reaction temperature (4 °C) have been chosen. Calcium carbonate polymorphs nucleate in the confinement of nanosized pores (⌀ 3–5 nm) of the S-layer. The hydrous crystalline calcium carbonate (ikaite) is formed initially with [110] as the favored growth direction. It transforms into the anhydrous metastable vaterite by a solid-state transition. In a following reaction step, calcite is precipitated, caused by dissolution of vaterite in the aqueous solution. In the larger pores of the cell wall (⌀ 20–50 nm), hydrated amorphous calcium carbonate is grown, which transforms into metastable monohydrocalcite, aragonite, or calcite. Due to the sequence of reaction steps via various metastable phases, the bacteria gain time for chipping the partially mineralized S-layer, and forming a fresh S-layer (characteristic growth time about 20 min). Thus, the bacteria can survive in solutions with high calcium carbonate supersaturation under the conditions of forced biomineralization.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

SUMMARYGalactose toxicity (Gal‐Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP‐galactose (UDP‐Gal) and UDP‐glucose is catalyzed by the enzymes galactokinase, UDP‐sugar pyrophosphorylase (USP) and UDP‐galactose 4‐epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose‐mediated toxicity in plants. In this study, we investigated the role of galactose‐containing metabolites like galactose‐1‐phosphate (Gal‐1P) and UDP‐Gal in Gal‐Tox. Recently published data from mouse models suggest that a reduction of the Gal‐1P level via an mRNA‐based therapy helps to overcome Gal‐Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal‐1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L−1 Gal). Nucleotide sugar analysis via high‐performance liquid chromatography‐mass spectrometry revealed highly elevated UDP‐Gal levels in roots of seedlings grown on 1.5 mmol L−1 galactose versus 1.5 mmol L−1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal‐feeding conditions, indicating that glycoproteins are a major target for elevated UDP‐Gal levels in plants.

Delivery of DNA into Human Cells by Functionalized Lignin Nanoparticles.

Lignin is an aromatic plant cell wall polymer that is generated in large quantities as a low-value by-product by the pulp and paper industry and by biorefineries that produce renewable fuels and chemicals from plant biomass. Lignin structure varies among plant species and as a function of the method used for its extraction from plant biomass. We first explored the impact of this variation on the physico-chemical properties of lignin nanoparticles (LNPs) produced via a solvent exchange procedure and then examined whether LNPs produced from industrial sources of lignin could be used as delivery vehicles for DNA. Spherical LNPs were formed from birch and wheat BioLignin™ and from poplar thioglycolic acid lignin after dissolving the lignin in tetrahydrofuran (THF) and dialyzing it against water. Dynamic light scattering indicated that the diameter of these LNPs was dependent on the initial concentration of the lignin, while electrophoretic light scattering indicated that the LNPs had a negative zeta potential, which became less negative as the diameter increased. The dynamics of LNP formation as a function of the initial lignin concentration varied as a function of the source of the lignin, as did the absolute value of the zeta potential. After coating the LNPs with cationic poly-l-lysine, an electrophoretic mobility shift assay indicated that DNA could adsorb to LNPs. Upon transfection of human A549 lung carcinoma basal epithelial cells with functionalized LNPs carrying plasmid DNA encoding the enhanced green fluorescent protein (eGFP), green foci were observed under the microscope, and the presence of eGFP in the transfected cells was confirmed by ELISA. The low cytotoxicity of these LNPs and the ability to tailor diameter and zeta potential make these LNPs of interest for future gene therapy applications.