Cell Wall Characteristics Research Articles

Visualization of Plant Cell Wall Epitopes Using Immunogold Labeling for Electron Microscopy.

Plant cell walls consist of different polysaccharides and structural proteins, which form a rigid layer located outside of the plasma membrane. The wall is also a very dynamic cell composite, which is characterized by complex polysaccharide interactions and various modifications during cell development. The visualization of cell wall components in situ is very challenging due to the small size of cell wall composites (nanometer scale), large diversity of the wall polysaccharides and their complex interactions. This protocol describes immunogold labeling of different cell wall epitopes for high-resolution transmission electron microscopy (TEM). It provides a detailed procedure for collection and preparation of plant material, ultra-thin sectioning, specimen labeling and contrasting. An immunolabeling procedure workflow was optimized to obtain high efficiency of carbohydrates labeling for high-resolution TEM. This method was applied to study plant cell wall characteristics in various plant tissues but could also be applied for other cell components in plant and animal tissues.

Fractionation and characterization of lignin streams from unique high-lignin content endocarp feedstocks

BackgroundLignin is a promising source of building blocks for upgrading to valuable aromatic chemicals and materials. Endocarp biomass represents a non-edible crop residue in an existing agricultural setting which cannot be used as animal feed nor soil amendment. With significantly higher lignin content and bulk energy density, endocarps have significant advantages to be converted into both biofuel and bioproducts as compared to other biomass resources. Deep eutectic solvent (DES) is highly effective in fractionating lignin from a variety of biomass feedstocks with high yield and purity while at lower cost comparing to certain ionic liquids.ResultsIn the present study, the structural and compositional features of peach and walnut endocarp cells were characterized. Compared to typical woody and herbaceous biomass, endocarp biomass exhibits significantly higher bulk density and hardness due to its high cellular density. The sugar yields of DES (1:2 choline chloride: lactic acid) pretreated peach pit (Prunus persica) and walnut shell (Juglans nigra) were determined and the impacts of DES pretreatment on the physical and chemical properties of extracted lignin were characterized. Enzymatic saccharification of DES pretreated walnut and peach endocarps gave high glucose yields (over 90%); meanwhile, compared with dilute acid and alkaline pretreatment, DES pretreatment led to significantly higher lignin removal (64.3% and 70.2% for walnut and peach endocarps, respectively). The molecular weights of the extracted lignin from DES pretreated endocarp biomass were significantly reduced. 1H–13C HSQC NMR results demonstrate that the native endocarp lignins were SGH type lignins with dominant G-unit (86.7% and 80.5% for walnut and peach endocarps lignins, respectively). DES pretreatment decreased the S and H-unit while led to an increase in condensed G-units, which may contribute to a higher thermal stability of the isolated lignin. Nearly all β-O-4′ and a large portion of β-5′ linkages were removed during DES pretreatment.ConclusionsThe high lignin content endocarps have unique cell wall characteristics when compared to the other lignocellulosic biomass feedstocks. DES pretreatment was highly effective in fractionating high lignin content endocarps to produce both sugar and lignin streams while the DES extracted lignins underwent significant changes in SGH ratio, interunit linkages, and molecular sizes.

A novel FC17/CESA4 mutation causes increased biomass saccharification and lodging resistance by remodeling cell wall in rice

BackgroundRice not only produces grains for human beings, but also provides large amounts of lignocellulose residues, which recently highlighted as feedstock for biofuel production. Genetic modification of plant cell walls can potentially enhance biomass saccharification; however, it remains a challenge to maintain a normal growth with enhanced lodging resistance in rice.ResultsIn this study, rice (Oryza sativa) mutant fc17, which harbors the substitution (F426S) at the plant-conserved region (P-CR) of cellulose synthase 4 (CESA4) protein, exhibited slightly affected plant growth and 17% higher lodging resistance compared to the wild-type. More importantly, the mutant showed a 1.68-fold enhancement in biomass saccharification efficiency. Cell wall composition analysis showed a reduction in secondary wall thickness and cellulose content, and compensatory increase in hemicelluloses and lignin content. Both X-ray diffraction and calcofluor staining demonstrated a significant reduction in cellulose crystallinity, which should be a key factor for its high saccharification. Proteomic profiling of wild-type and fc17 plants further indicated a possible mechanism by which mutation induces cellulose deposition and cell wall remodeling.ConclusionThese results suggest that CESA4 P-CR site mutation affects cell wall features especially cellulose structure and thereby causes enhancement in biomass digestion and lodging resistance. Therefore, CESA4 P-CR region is promising target for cell wall modification to facilitate the breeding of bioenergy rice.

Characterization of compensatory mutations associated with restoration of daptomycin-susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus and the role mprF mutations.

The objective of this study was to investigate the underlying mechanism explaining reversion of clinical DAP non-susceptible (NS) MRSA isolates to DAP-susceptible (S) by analysis of genomic and cell wall characteristics of clinical DAP-NS MRSA and DAP-S MRSA isolates as well as invitro revertant DAP-S MRSA using whole genome sequencing (WGS) and analysis of biological properties. WGS of the 4 clinical DAP-NS MRSA revealed mprF mutations resulting in amino acid substitutions or deletion. These same amino acid substitutions and deletion were also observed in the 4 invitro revertant DAP-S strains. While WGS identified the presence of the same mprF mutations in both the DAP-NS and invitro DAP-S revertant strains, new mutations were also detected in other genes and intergenic regions of invitro DAP-S revertant strains. Transmission electron microscopy to assess cell-wall (CW) thickness of 4 sets strains (pre- and post-DAP therapy isolates and invitro DAP-S revertant) showed that 3 of the 4 isolates developed increased thickness of the CW after DAP therapy. After reversion to DAP susceptibility, CW thickness was decreased to the same level as DAP-S MRSA. Our results indicate that invitro conversion of DAP-NS MRSA to DAP-S is independent of mprF gene mutations and may be partially explained by a change in CW thickness. However, as some strains showed no change in the CW, further studies are required to elucidate the different mechanisms of resistance to DAP, and factors for conversion of DAP-NS to DAP-S.

Complete substitution of a secondary cell wall with a primary cell wall in Arabidopsis

The bulk of a plant's biomass, termed secondary cell walls, accumulates in woody xylem tissues and is largely recalcitrant to biochemical degradation and saccharification1. By contrast, primary cell walls, which are chemically distinct, flexible and generally unlignified2, are easier to deconstruct. Thus, engineering certain primary wall characteristics into xylem secondary walls would be interesting to readily exploit biomass for industrial processing. Here, we demonstrated that by expressing AP2/ERF transcription factors from group IIId and IIIe in xylem fibre cells of mutants lacking secondary walls, we could generate plants with thickened cell wall characteristics of primary cell walls in the place of secondary cell walls. These unique, newly formed walls displayed physicochemical and ultrastructural features consistent with primary walls and had gene expression profiles illustrative of primary wall synthesis. These data indicate that the group IIId and IIIe AP2/ERFs are transcription factors regulating primary cell wall deposition and could form the foundation for exchanging one cell wall type for another in plants.

In situ deep eutectic solvent pretreatment to improve lignin removal from garden wastes and enhance production of bio-methane and microbial lipids

Biomass pretreatment can improve the conversion efficiency of bioenergy production. Liquid hot water (LHW) pretreatment is a truly green pretreatment due to its zero chemical use, but has the disadvantages of low lignin removal and pseudo-lignin formation. A modified liquid hot water (MLHW) process based on in situ synthesis of deep eutectic solvent (DES) could efficiently improve delignification of Roystonea regia leaves (LR) and leaf sheaths (LSR). LSR was less recalcitrant than LR, and its characteristics of higher porosity (34.8%) and thinner cell walls (5.2 μm) for LSR contributed it higher lignin removal (53.6%) and lower choline chloride (ChCl) consumption (H2O-ChCl mass ratio of 2:1) than those (44.6% and 1:2) from LR. Moreover, a great improvement of 309.0% in bio-methane yield was achieved for the MLHW-treated LSR. In addition, in situ DES in MLHW had good biocompatibility with cellulase, microalgae, and yeast.

Prediction of indigestible NDF in South African and Australian forages from cell wall characteristics

A portion of the forage cell wall is unavailable to microbial digestion in ruminants and in previous work this fraction was characterized by the relationship between lignin (sa) and neutral detergent fiber (NDF) as 2.4 × lignin and termed the indigestible NDF (iNDF). Further, this relationship was considered to be a static relationship between lignin (sa) and NDF with no genetic variation or observed effects of growing conditions on the plant. The objectives of this work were to further characterize the relationship between lignin and iNDF and to evaluate the concept of a fixed value, assuming that the relationship is more dynamic and dependent on agronomic and environmental factors. A further objective was to predict iNDF from cell wall composition. More than two hundred samples of several forage species from Australia and South Africa were analyzed for NDF, acid detergent fiber, lignin and iNDF. Forages were classified according to origins, type and species. The ratios of iNDF to ADL ranged between 1.6 and 8.0, demonstrating the dynamic nature of the relationship. Further, an attempt to predict iNDF based on the relationship between ADL/NDF and the ratio was made and the equations provided R2 values between 0.82 and 0.95, indicating that a prediction is possible, especially when considering one species at a time and more information within forage group and single forages on agronomic and environmental conditions would explain the extra variation. A single value of the ratio iNDF to ADL does not allow proper estimation of the size of the potentially digestible NDF and its rate of digestion, but species-specific equations can be developed and be effective in predicting iNDF. Long-term fermentations should however be preferred to correctly quantify these values and, if not available, data should allow for specific equations or near infrared spectroscopy calibration equations.

A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

BackgroundIn flowering plants, the male gametophyte (pollen) is one of the most vulnerable cells to temperature stress. In Arabidopsis thaliana, a pollen-specific CyclicNucleotide-Gated cationChannel 16 (cngc16), is required for plant reproduction under temperature-stress conditions. Plants harboring a cncg16 knockout are nearly sterile under conditions of hot days and cold nights. To understand the underlying cause, RNA-Seq was used to compare the pollen transcriptomes of wild type (WT) and cngc16 under normal and heat stress (HS) conditions.ResultsHere we show that a heat-stress response (HSR) in WT pollen resulted in 2102 statistically significant transcriptome changes (≥ 2-fold changes with adjusted p-value ≤0.01), representing approximately 15% of 14,226 quantified transcripts. Of these changes, 89 corresponded to transcription factors, with 27 showing a preferential expression in pollen over seedling tissues. In contrast to WT, cngc16 pollen showed 1.9-fold more HS-dependent changes (3936 total, with 2776 differences between WT and cngc16). In a quantitative direct comparison between WT and cngc16 transcriptomes, the number of statistically significant differences increased from 21 pre-existing differences under normal conditions to 192 differences under HS. Of the 20 HS-dependent changes in WT that were most different in cngc16, half corresponded to genes encoding proteins predicted to impact cell wall features or membrane dynamics.ConclusionsResults here define an extensive HS-dependent reprogramming of approximately 15% of the WT pollen transcriptome, and identify at least 27 transcription factor changes that could provide unique contributions to a pollen HSR. The number of statistically significant transcriptome differences between WT and cngc16 increased by more than 9-fold under HS, with most of the largest magnitude changes having the potential to specifically impact cell walls or membrane dynamics, and thereby potentiate cngc16 pollen to be hypersensitive to HS. However, HS-hypersensitivity could also be caused by the extensive number of differences throughout the transcriptome having a cumulative effect on multiple cellular pathways required for tip growth and fertilization. Regardless, results here support a model in which a functional HS-dependent reprogramming of the pollen transcriptome requires a specific calcium-permeable Cyclic Nucleotide-Gated cation Channel, CNGC16.

Cytological differentiation and cell wall involvement in the growth mechanisms of articulated laticifers in Tabernaemontana catharinensis A.DC. (Apocynaceae).

The cellular mechanisms of laticifer growth are of particular interest in plant biology but are commonly neglected. Using transmission electron microscopy and immunocytochemical methods, we recorded cytological differentiation and evaluated the cell wall involvement in the growth of articulated laticifers with intrusive growth in the mature embryo and plant shoot apex of Tabernaemontana catharinensis. The incorporation of adjacent meristematic cells into the laticifer system occurred in the embryo and plant shoot apex, and the incorporated cells acquired features of laticifer, confirming the laticifers' action-inducing mechanism. In the embryo, this was the main growth mechanism, and began with enlargement of the plasmodesmata and the formation of pores between laticifers and meristematic cells. In the plant shoot apex, it began with loose and disassembled walls and the reorientation of the cortical microtubules of the incorporated cell. Plasmodesmata were absent in these laticifers. There was stronger evidence of intrusive growth in undifferentiated portions of the plant shoot apex than in the embryo. The numerous plasmodesmata in laticifers of the embryo may have been related to the lower frequency of intrusive growth. Intrusive growth was associated with presence of arabinan (increasing wall flexibility and fluidity), and absence of galactan (avoiding wall stiffness), and callose (as a consequence of reduction in symplastic connections) in the laticifer walls. The abundance of low de-methyl-esterified homogalacturonan in the middle lamella and corners may reestablish cell-cell bonding in the laticifers. The cell wall features differed between embryo and plant shoot apex and are directly associated to laticifer growth mechanisms.

Broad spectrum developmental role of Brachypodium AUX1.

Summary Targeted cellular auxin distribution is required for morphogenesis and adaptive responses of plant organs. In Arabidopsis thaliana (Arabidopsis), this involves the prototypical auxin influx facilitator AUX1 and its LIKE‐AUX1 (LAX) homologs, which act partially redundantly in various developmental processes. Interestingly, AUX1 and its homologs are not strictly essential for the Arabidopsis life cycle. Indeed, aux1 lax1 lax2 lax3 quadruple knock‐outs are mostly viable and fertile, and strong phenotypes are only observed at low penetrance.Here we investigated the Brachypodium distachyon (Brachypodium) AUX1 homolog BdAUX1 by genetic, cell biological and physiological analyses.We report that BdAUX1 is essential for Brachypodium development. Bdaux1 loss‐of‐function mutants are dwarfs with aberrant flower development, and consequently infertile. Moreover, they display a counter‐intuitive root phenotype. Although Bdaux1 roots are agravitropic as expected, in contrast to Arabidopsis aux1 mutants they are dramatically longer than wild type roots because of exaggerated cell elongation. Interestingly, this correlates with higher free auxin content in Bdaux1 roots. Consistently, their cell wall characteristics and transcriptome signature largely phenocopy other Brachypodium mutants with increased root auxin content.Our results imply fundamentally different wiring of auxin transport in Brachypodium roots and reveal an essential role of BdAUX1 in a broad spectrum of developmental processes, suggesting a central role for AUX1 in pooideae.

Genetic Engineering of Energy Crops to Reduce Recalcitrance and Enhance Biomass Digestibility

Bioenergy, biofuels, and a range of valuable chemicals may be extracted from the abundantly available lignocellulosic biomass. To reduce the recalcitrance imposed by the complex cell wall structure, genetic engineering has been proposed over the years as a suitable solution to modify the genes, thereby, controlling the overall phenotypic expression. The present review provides a brief description of the plant cell wall structure and its compositional array i.e., lignin, cellulose, hemicellulose, wall proteins, and pectin, along with their effect on biomass digestibility. Also, this review discusses the potential to increase biomass by gene modification. Furthermore, the review highlights the potential genes associated with the regulation of cell wall structure, which can be targeted for achieving energy crops with desired phenotypes. These genetic approaches provide a robust and assured method to bring about the desired modifications in cell wall structure, composition, and characteristics. Ultimately, these genetic modifications pave the way for achieving enhanced biomass yield and enzymatic digestibility of energy crops, which is crucial for maximizing the outcomes of energy crop breeding and biorefinery applications.

Cell wall features transferred from common into durum wheat to improve Fusarium Head Blight resistance

Durum wheat is naturally more susceptible to Fusarium graminerum infection in comparison to common wheat. The improvement of durum wheat resistance against F. graminearum is a challenge due to the lack of resistance sources in its gene pool. FHB-resistance factors were introduced in durum wheat by generating recombinant inbred lines (RILs), obtained by crossing the hexaploid resistant accession 02-5B-318 with the susceptible durum wheat cv. Saragolla. In this work we explored the possible contribution of cell wall (CW) in RILs with improved FHB resistance. We thoroughly studied CW components, mycotoxins content and the expression of related genes in different RILs selected for their extremely high and low resistance to FHB. Differences were found in resistant and susceptible lines in the degree of pectin methylesterification and in deoxynivalenol (DON) accumulation after fungal infection. Genes involved in biochemical modification of CW structure (WheatPme-1, Glu-1) and mycotoxins accumulation (ns-LTP-1) were analyzed as putative candidates for FHB resistance. Our results indicate that durum wheat plants with cell wall structure and gene response acquired from common wheat displayed an increased resistance to FHB.

A comprehensive review on pretreatment of microalgae for biogas production

Use of microalgal biomass for renewable energy production has gained considerable attention in the world due to increasing global energy demand and negative environmental impacts of nonrenewable fossil fuels. Anaerobic digestion is one of the renewable technologies that microalgal biomass is converted into biogas by anaerobic archea. One of the main drawbacks of using microalgal biomass for biogas production is that certain types of microalgae has rigid cell wall characteristics, which limits accessibility of anaerobic archea to microalgal intracellular organic matter during hydrolysis phase. This limitation lowers efficiency of biogas production from microalgal biomass. However, introducing pretreatment methods prior to anaerobic digestion provides disruption of rigid microalgal cell wall and improve biogas yields from microalgal biomass. The objective of this paper was to review current knowledge related to pretreatment methods applied prior to anaerobic digestion of microalgal biomass. Efficiency and applicability of pretreatment methods mainly depend on type of microalgae, cell wall characteristics, and cost and energy requirements during pretreatment process. In this review, various type of pretreatment methods applied to microalgal biomass was discussed in detail with background knowledge and literature studies in their potential on maximization of biogas yields and their cost effectiveness, which is important for large-scale applications. In the view of current knowledge, it was concluded that each pretreatment method has a relative contribution to improvement in biogas production depending on the type of microalgae. However, energy and cost requirements are the main limitations for pretreatment. So, further studies should focus on reduction of cost and energy demand by introducing combined methods, novel chemicals, and on-site or immobilized enzymes in pretreatment to increase feasibility of pretreatment prior to anaerobic digestion in industrial scale.

Changes in photosynthetic rate and stress volatile emissions through desiccation-rehydration cycles in desiccation-tolerant epiphytic filmy ferns (Hymenophyllaceae).

Exposure to recurrent desiccation cycles carries a risk of accumulation of reactive oxygen species that can impair leaf physiological activity upon rehydration, but changes in filmy fern stress status through desiccation and rewatering cycles have been poorly studied. We studied foliage photosynthetic rate and volatile marker compounds characterizing cell wall modifications (methanol) and stress development (lipoxygenase [LOX] pathway volatiles and methanol) through desiccation-rewatering cycles in lower-canopy species Hymenoglossum cruentum and Hymenophyllum caudiculatum, lower- to upper-canopy species Hymenophyllumplicatum and upper-canopy species Hymenophyllum dentatum sampled from a common environment and hypothesized that lower canopy species respond more strongly to desiccation and rewatering. In all species, rates of photosynthesis and LOX volatile emission decreased with progression of desiccation, but LOX emission decreased with a slower rate than photosynthesis. Rewatering first led to an emission burst of LOX volatiles followed by methanol, indicating that the oxidative burst was elicited in the symplast and further propagated to cell walls. Changes in LOX emissions were more pronounced in the upper-canopy species that had a greater photosynthetic activity and likely a greater rate of production of photooxidants. We conclude that rewatering induces the most severe stress in filmy ferns, especially in the upper canopy species.

A broadly conserved NERD genetically interacts with the exocyst to affect root growth and cell expansion.

The exocyst, a conserved, octameric protein complex, helps mediate secretion at the plasma membrane, facilitating specific developmental processes that include control of root meristem size, cell elongation, and tip growth. A genetic screen for second-site enhancers in Arabidopsis identified NEW ENHANCER of ROOT DWARFISM1 (NERD1) as an exocyst interactor. Mutations in NERD1 combined with weak exocyst mutations in SEC8 and EXO70A1 result in a synergistic reduction in root growth. Alone, nerd1 alleles modestly reduce primary root growth, both by shortening the root meristem and by reducing cell elongation, but also result in a slight increase in root hair length, bulging, and rupture. NERD1 was identified molecularly as At3g51050, which encodes a transmembrane protein of unknown function that is broadly conserved throughout the Archaeplastida. A functional NERD1-GFP fusion localizes to the Golgi, in a pattern distinct from the plasma membrane-localized exocyst, arguing against a direct NERD1-exocyst interaction. Structural modeling suggests the majority of the protein is positioned in the lumen, in a β-propeller-like structure that has some similarity to proteins that bind polysaccharides. We suggest that NERD1 interacts with the exocyst indirectly, possibly affecting polysaccharides destined for the cell wall, and influencing cell wall characteristics in a developmentally distinct manner.

Farklı bölgelerden alınan Kızılçam (Pinus Brutia Ten.) odunlarının bazı anatomik, kimyasal ve lifsel özelliklerinin karşılaştırılması

Aim of study: In this study, it was aimed to research the anatomical properties, chemical composition of the cell wall and fiber characteristics of Turkish Pine (Pinus brutia Ten.) wood sampled from five different zone of Kastamonu province in Turkey. Area of study: This study was performed at the laboratories of Department of Forest Industry Engineering and Department of Biology, Kastamonu University. Material and Methods: Turkish Pine (Pinus brutia Ten.) wood samples obtained from five different zone of Kastamonu (Hanönü, Bozkurt, Araç, İnebolu and Tosya) were used as experimental materials. Anatomical properties (ray number, tracheid widths, widths of early and late wood zones), wood chemical components (ethanol extraction (%), lignin (%), holocellulose (%), alpha cellulose (%), 1% NaOH solubility (%), cold water solubility (%), hot water solubility (%) and ash specification (%)) and fibrous characteristics of wood fibers (fiber length, fiber width, lumen width and cell wall thickness) were measured according to standard methods.Main results: This study indicated that the samples obtained from Tosya have maximum fibre length with 3.15 mm and maximum lumen width with 27.75 mm, whereas maximum fibre width was found in samples obtained from İnebolu with 42.27 mm and maximum cell wall thickness was seen in samples of İnebolu with 7.90 mm. As a consequence of chemical analyses, it was confirmed that holocellulose ratio, alpha-cellulose ratio, lignin ratio, 1% NaOH solubility, ethanol solubility, hot water solubility, cold water solubility and ash ratio changed within the range of 70.37%-72.38%, 42.62%-47.30%, 26.04%-27.86%, 12.38%-13.14%, 2.38%-3.41%, 2.86%-3.83%, 2.29%-3.27% and 0.26%-0.40%, respectively. When it was looked to the results of anatomical research, it was determined that the samples taken from Hanönü had maximum average ray numbers with 7. Likewise, maximum tracheid widths were found samples in İnebolu and Bozkurt with 1.31 µm. Besides, maximum earlywood width was confirmed samples taken from Bozkurt with 0.47 mm, whereas maximum latewood width was confirmed samples taken from Hanönü with 1.31 mm. Research highlights: It is inferred that the same tree species taken from different areas have varied anatomical, chemical and fibre properties measured by means of this study. Because of this, it can be stated that growth location properties and growth media affect the characteristics of same type trees.

Steam Explosion and Vibrating Membrane Filtration to Improve the Processing Cost of Microalgae Cell Disruption and Fractionation

The aim of this study is to explore an innovative downstream route for microalgae processing to reduce cost production. Experiments have been carried out on cell disruption and fractionation stages to recover lipids, sugars, and proteins. Steam explosion and dynamic membrane filtration were used as unit operations. The species tested were Nannochloropsis gaditana, Chlorella sorokiniana, and Dunaliella tertiolecta with different cell wall characteristics. Acid-catalysed steam explosion permitted cell disruption, as well as the hydrolysis of carbohydrates and partial hydrolysis of proteins. This permitted a better access to non-polar solvents for lipid extraction. Dynamic filtration was used to moderate the impact of fouling. Filtration enabled two streams: A permeate containing water and monosaccharides and a low-volume retentate containing the lipids and proteins. The necessary volume of solvent to extract the lipids is thus much lower. An estimation of operational costs of both steam explosion and membrane filtration was performed. The results show that the steam explosion operation cost varies between 0.005 $/kg and 0.014 $/kg of microalgae dry sample, depending on the cost of fuel. Membrane filtration cost in fractionation was estimated at 0.12 $/kg of microalgae dry sample.

From nano- to micrometer scale: the role of microwave-assisted acid and alkali pretreatments in the sugarcane biomass structure

BackgroundTo date, great strides have been made in elucidating the role of thermochemical pretreatments in the chemical and structural features of plant cell walls; however, there is no clear picture of the plant recalcitrance and its relationship to deconstruction. Previous studies precluded full answers due to the challenge of multiscale features of plant cell wall organization. Complementing the previous efforts, we undertook a systematic, multiscale, and integrated approach to track the effect of microwave-assisted H2SO4 and NaOH treatments on the hierarchical structure of plants, i.e., from a nano- to micrometer scale. We focused on the investigation of the highly recalcitrant sclerenchyma cell walls from sugarcane bagasse.ResultsThrough atomic force microscopy and X-ray diffraction analyses, remarkable details of the assembly of cellulose microfibrils not previously seen were revealed. Following the H2SO4 treatment, we observed that cellulose microfibrils were almost double the width of the alkali pretreated sample at the temperature of 160 °C. Such enlargement led to a greater contact between cellulose chains, with a subsequent molecule alignment, as indicated by the X-ray diffraction (XRD) results with the conspicuous expansion of the average crystallite size. The delignification process had little effect on the local nanometer-sized arrangement of cellulose molecules. However, the rigidity and parallel alignment of cellulose microfibrils were partially degraded. The XRD analysis also agrees with these findings as evidenced by large momentum transfer vectors (q > 20 nm−1), interpreted as indicators of the long-range order of cell wall components, which were similar for all the studied samples except with application of the NaOH treatment at 160 °C. These changes were followed by the eventual swelling of the fiber cell walls.ConclusionsBased on an integrated approach, we presented multidimensional architectural models of cell wall deconstruction resulting from microwave-assisted pretreatments. We provided direct evidence supporting the idea that hemicellulose is the main barrier for the swelling of cellulose microfibrils, whereas lignin adds rigidity to cell walls. Our findings shed light on the design of more efficient strategies, not only for the conversion of biomass to fuels but also for the production of nanocellulose, which has great potential for several applications such as composites, rheology modifiers, and pharmaceuticals.

Unveiling 3D physicochemical changes of sugarcane bagasse during sequential acid/alkali pretreatments by synchrotron phase-contrast imaging

Several pretreatment strategies focus on the removal of a significant part of lignin from plant cell walls, as it is considered the main barrier to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The ability to chemically differentiate and spatially locate lignins across cell walls provides an important contribution to the effort to improve these processes. Here, we present a novel approach using synchrotron phase-contrast tomography to probe the physicochemical features of plant cell walls. In addition to the 3D cellular architecture, the distribution of dense packed lignin–carbohydrate complexes (or, simply dense lignin) over larger regions and within cell walls has been successfully provided. We examined in particular the effect of the sequential H2SO4 and NaOH pretreatment on sugacane bagasse. Our results revealed that aggregates of dense lignin form elongated 3D structures in cell corners (CC) following the orientation of the sclerenchyma fibers; a remarkable result in the light of the conventional perception that lignin particles are discrete and roughly spherical. After the acid pretreatment, a considerable fraction of the dense lignin primarily located in CCs was removed; and it was further dissolved with the subsequent alkali treatment. Unexpectedly, the two-step pretreatment did not contribute to the cellulose accessibility in terms of available surface area and porosity. This study provides new insights into the underlying mechanisms of biomass deconstruction by pretreatments; and the approach established here can be extended to other systems relevant to the bioenergy and biotechnology arenas.

Stereochemical identification of glucans by oligothiophenes enables cellulose anatomical mapping in plant tissues

Efficient use of plant-derived materials requires enabling technologies for non-disruptive composition analysis. The ability to identify and spatially locate polysaccharides in native plant tissues is difficult but essential. Here, we develop an optical method for cellulose identification using the structure-responsive, heptameric oligothiophene h-FTAA as molecular fluorophore. Spectrophotometric analysis of h-FTAA interacting with closely related glucans revealed an exceptional specificity for β-linked glucans. This optical, non-disruptive method for stereochemical differentiation of glycosidic linkages was next used for in situ composition analysis in plants. Multi-laser/multi-detector analysis developed herein revealed spatial localization of cellulose and structural cell wall features such as plasmodesmata and perforated sieve plates of the phloem. Simultaneous imaging of intrinsically fluorescent components revealed the spatial relationship between cell walls and other organelles, such as chloroplasts and lignified annular thickenings of the trachea, with precision at the sub-cellular scale. Our non-destructive method for cellulose identification lays the foundation for the emergence of anatomical maps of the chemical constituents in plant tissues. This rapid and versatile method will likely benefit the plant science research fields and may serve the biorefinery industry as reporter for feedstock optimization as well as in-line monitoring of cellulose reactions during standard operations.