Cell Wall Chemistry Research Articles

Cell wall chemistry and tissue structure underlie shifts in material properties of a perennial kelp

ABSTRACTLaminaria is an abundant kelp genus in temperate nearshore ecosystems that grows with a circannual ‘stop-start’ pattern. Species of Laminaria play important ecological roles in kelp forests worldwide and are harvested commercially as a source of food and valuable extracts. In order to evaluate seasonal differences in tissue properties and composition, we compared the material properties, histology and cell-wall composition of overwintering blades with newly synthesized, actively growing blades from Laminaria setchellii. We found that overwintering blades were fortified with a thicker cortex and increased cell wall investment, leading to increased material strength. Overwintering tissues were composed of higher proportions of cellulose and fucose-containing polysaccharides (i.e. FCSPs, fucoidans) than newly formed blades and were found to possess thicker cell walls, likely to withstand the waves of winter storms. Chemical cell wall profiling revealed that significant proportions of fucose were associated with cellulose, especially in overwintering tissues, confirming the association between cellulose and some fucose-containing polysaccharides. Changes in material properties during the resting phase may allow these kelps to retain their non-growing blades through several months of winter storms. The results of this study demonstrate how one species might regulate its material properties seasonally, and at the same time shed light on the mechanisms that might control the material properties of kelps in general.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula×tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Selective precipitation and characterization of lignin-carbohydrate complexes (LCCs) from Eucalyptus.

Six types of lignin-carbohydrate complex (LCC) fractions were isolated from Eucalyptus. The acidic dioxane treatment applied significantly improved the yield of LCCs. The extraction conditions had a limited impact on the LCC structures and linkages. Characterization of the lignin-carbohydrate complex (LCC) structures and linkages promises to offer insight on plant cell wall chemistry. In this case, Eucalyptus LCCs were extracted by aqueous dioxane, and then precipitated sequentially by 70% ethanol, 100% ethanol, and acidic water (pH=2). The composition and structure of the six LCC fractions obtained by selective precipitation were investigated by sugar analysis, molecular weight determination, and 2D HSQC NMR. It was found that the acidic (0.05-M HCl) dioxane treatment significantly improved the yield of LCCs (66.4% based on Klason lignin), which was higher than the neutral aqueous dioxane extraction, and the extraction condition showed limited impact on the LCC structures and linkages. In the fractionation process, the low-molecular-weight LCCs containing a high content of carbohydrates (60.3-63.2%) were first precipitated by 70% ethanol from the extractable solution. The phenyl glycoside (PhGlc) bonds (13.0-17.0 per 100Ar) and highly acetylated xylans were observed in the fractions recovered by the precipitation with 100% ethanol. On the other hand, such xylan-rich LCCs exhibited the highest frequency of β-O-4 linkages. The benzyl ether (BE) bonds were only detected in the fractions obtained by acidic water precipitation.

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging.

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.

PG-Metrics: A chemometric-based approach for classifying bacterial peptidoglycan data sets and uncovering their subjacent chemical variability.

Bacteria cells are protected from osmotic and environmental stresses by an exoskeleton-like polymeric structure called peptidoglycan (PG) or murein sacculus. This structure is fundamental for bacteria’s viability and thus, the mechanisms underlying cell wall assembly and how it is modulated serve as targets for many of our most successful antibiotics. Therefore, it is now more important than ever to understand the genetics and structural chemistry of the bacterial cell walls in order to find new and effective methods of blocking it for the treatment of disease. In the last decades, liquid chromatography and mass spectrometry have been demonstrated to provide the required resolution and sensitivity to characterize the fine chemical structure of PG. However, the large volume of data sets that can be produced by these instruments today are difficult to handle without a proper data analysis workflow. Here, we present PG-metrics, a chemometric based pipeline that allows fast and easy classification of bacteria according to their muropeptide chromatographic profiles and identification of the subjacent PG chemical variability between e.g. bacterial species, growth conditions and, mutant libraries. The pipeline is successfully validated here using PG samples from different bacterial species and mutants in cell wall proteins. The obtained results clearly demonstrated that PG-metrics pipeline is a valuable bioanalytical tool that can lead us to cell wall classification and biomarker discovery.

Parenchyma cell wall structure in twining stem of Dioscorea balcanica

Anatomical adaptation of liana plants includes structural changes in cell walls of different tissues: fibers, vessel elements and tracheids. However, the contribution of parenchyma cells to stem twining in liana plants is mostly unknown. The aim of this investigation is to determine changes in stem parenchyma cell walls that are correlated with the twinning process in liana plants. Parenchyma cell wall structure was studied on the stem cross sections of straight and twisted internodes of monocotyledonous liana Dioscorea balcanica, by different microscopy techniques: light microscopy, scanning electron microscopy, fluorescence detected linear dichroism microscopy and Fourier transform infrared microspectrometry. In addition, chemical analysis of the entire stem internodes was performed using photometric and chromatographic methods. Parenchyma cell walls of twisted D. balcanica internodes are characterized by: lower amounts of cellulose (obtained by FTIR microspectrometry) with different cellulose microfibril orientation (shown by Scanning electron microscopy), but no changes in “cellulose fibril order” (obtained by Differential polarization laser scanning microscopy); lower amounts of xyloglucan, higher amounts of xylan, higher amounts of lignin with modified organization—less condensed lignin (obtained by FTIR microspectrometry). At the same time, chemical analysis of the entire internodes did not show significant differences in lignin content and cell wall bound phenols related to stem twining, except for the presence of diferulate cross-links exclusively in twisted internodes. Our results indicate that adaptations to mechanical strain in D. balcanica stems involve modifications in parenchyma cell wall structure and chemistry, which provide decreased stiffness, higher strength and increased elasticity of twisted internodes.

Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility.

SummarySustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate‐expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.

Immunolocalization of pectin and hemicellulose epitopes in the phloem of Norway spruce and Scots pine

Key messageDistribution patterns of pectin and hemicellulose epitopes in the phloem of Pinaceae conifers differ between cell types including sieve cells, axial/ray parenchyma cells and stone cells, which is more prominent in seedlings than mature trees.Although there is considerable information on the gross chemistry of conifer bark, little is known on the chemistry of secondary phloem at the individual cell level. This study investigated distribution of pectins and hemicelluloses in the phloem of two conifer species (Norway spruce and Scots pine) at an individual cell wall level using nine monoclonal antibodies specific for pectin and hemicellulose epitopes combined with immunofluorescence and TEM immunogold labeling. Differences in phloem cell wall chemistry between juvenile (seedlings) and mature conifer trees were also examined. The two conifer species showed qualitatively similar distribution patterns of epitopes in sieve- and (axial/ray) parenchyma cells, irrespective of seedlings and mature trees. Sieve- and parenchyma cell walls showed the presence of rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and xyloglucan epitopes, but revealed the absence of heteroxylan epitopes. Heteromannan epitopes were only detected in sieve cell walls, showing a chemical difference between sieve- and parenchyma cells. In contrast to qualitative similarity, there were several quantitative differences of epitope localization in sieve- and parenchyma cells between the two conifer species, indicating variations in the chemical structure and/or the amount of pectins and hemicelluloses between the two conifer species. These differences were more significant in seedlings than mature trees. Immunogold labeling of Norway spruce seedlings further indicated the possibility of chemical variations between cell wall regions within a single sieve cell wall. Phloem stone cells detected in mature Norway spruce showed the presence of heteromannans/heteroxylans and RG-I/HG/xyloglucans in secondary cell wall and middle lamellae, respectively.

Does copper tolerance provide a competitive advantage for degrading copper treated wood by soft rot fungi?

The ability of soft rot fungi possessing strong (Phialophora malorum), medium (Phialophora mutabilis) and poor copper tolerance (Chaetomium globosum) to degrade untreated and CuSO4 and micronized copper treated birch- and pine wood was assessed using ENV 807 standard tests. The aim was to determine whether an ability to grow on Cu-agar and copper in liquid cultures can be transcribed into a competitive advantage to degrade Cu-treated wood. An ability to tolerate high copper levels in-vitro was not correlated with increased decay by the fungi but rather reflected the native chemistry of the wood cell walls. Both untreated and Cu-treated wood were degraded by the three fungi and showed aggressiveness in the order C. globosum > P. mutabilis > P. malorum Higher mass loss was recorded for birch than pine and decreased progressively as the copper loadings increased with statistically insignificant difference noted between Cu-treatments. Microscopy showed decay at the cell wall level to reflect degree of lignification with parenchyma cells degraded first in both untreated and Cu-treated wood. Results indicate presence of copper and its toxicity is unlikely to be the main reason for preventing soft rot decay of wood but rather the additive effect of copper binding to the wood material.

Analyzing Cell Wall Elasticity After Hormone Treatment: An Example Using Tobacco BY-2 Cells and Auxin.

Atomic force microscopy, and related nano-indentation techniques, is a valuable tool for analyzing the elastic properties of plant cell walls as they relate to changes in cell wall chemistry, changes in development, and response to hormones. Within this chapter I will describe a method for analyzing the effect of the phytohormone auxin on the cell wall elasticity of tobacco BY-2 cells. This general method may be easily altered for different experimental systems and hormones of interest.

Microbiome and Exudates of the Root and Rhizosphere of Brachypodium distachyon, a Model for Wheat.

The rhizosphere microbiome is regulated by plant genotype, root exudates and environment. There is substantial interest in breeding and managing crops that host root microbial communities that increase productivity. The eudicot model species Arabidopsis has been used to investigate these processes, however a model for monocotyledons is also required. We characterized the rhizosphere microbiome and root exudates of Brachypodium distachyon, to develop it as a rhizosphere model for cereal species like wheat. The Brachypodium rhizosphere microbial community was dominated by Burkholderiales. However, these communities were also dependent on how tightly they were bound to roots, the root type they were associated with (nodal or seminal roots), and their location along the roots. Moreover, the functional gene categories detected in microorganisms isolated from around root tips differed from those isolated from bases of roots. The Brachypodium rhizosphere microbiota and root exudate profiles were similar to those reported for wheat rhizospheres, and different to Arabidopsis. The differences in root system development and cell wall chemistry between monocotyledons and eudicots may also influence the microorganism composition of these major plant types. Brachypodium is a promising model for investigating the microbiome of wheat.

VARIATIONS IN CELL WALL ULTRASTRUCTURE AND CHEMISTRY IN CELL TYPES OF EARLYWOOD AND LATEWOOD IN ENGLISH OAK (QUERCUS ROBUR)

Although there is considerable information on anatomy and gross chemistry of oak wood, little is known on the ultrastructure and chemistry at the individual cell wall level. In particular, differences in ultrastructure and chemistry within the same cell type between earlywood (EW) and latewood (LW) are poorly understood. This study investigated the ultrastructure and chemistry of (vasicentric) tracheids, vessels, (libriform) fibers and axial/ray parenchyma cells of English oak xylem (Quercus robur L.) using light-, fluorescence- and transmission electron microscopy combined with histo/cytochemistry and immunohisto/ cytochemistry. EW tracheids showed several differences from LW tracheids including thinner cell walls, wider middle lamella cell corner (MLcc) regions and lesser amounts of mannan epitopes. Fibers showed thicker cell walls and higher amounts of mannan epitopes than tracheids. EW vessels were rich in guaiacyl (G) lignin with a characteristic non-layered cell wall organization (absence of S1–3 layers), whereas LW vessels were rich in syringyl (S) lignin with a three layered cell wall structure (S1–3 layers). Formation of a highly lignified and wide protective layer (PL) inside axial/ray parenchyma cells was detected only in EW. Distribution of mannan epitopes varied greatly between cell types and between EW and LW, whereas distribution of xylan epitopes was almost identical in all cell types within a growth ring. Together, this study demonstrates that there are great variations in ultrastructure and chemistry of cell walls within a single growth ring of English oak xylem.

The effects of both insoluble lignin and the macromolecular traits of cellulose on the content of saccharides within solids during hydrothermal pretreatment of hybrid poplar wood

Liquid hot water pretreatment modifies the physiochemical features of plant cell walls, including cell wall chemistry and composition, as well as cellulose and lignin ultrastuctures. This study was designed to assess how the presence of insoluble lignin, lignin monomer composition, and the macromolecular traits of deconstructed cellulose influence the content of glucose, xylose and total saccharides within solids during liquid hot water pretreatment of hybrid poplar wood. Hydrothermal pretreatments at 180°C for both 5 and 10min were found to be the most effective conditions to achieve maximum glucose content. Under these pretreatment conditions, it was possible to achieve a more than 50% decrease in xylose content due to the solubilization of hemicelluloses, a low lignin content, and severe drops in the macromolecular traits of cellulose such as number-average molecular weight, weight-average molecular weight, degree of polymerization and polydispersity index. A continuous increase in the severity of the hydrothermal pretreatment resulted in a decrease in glucose content, an increase in insoluble lignin content, and an ongoing scission of cellulose chains. The amounts of glucose, xylose and total saccharides within solids were negatively influenced by the insoluble lignin content and the syringyl-to-guaiacyl ratio in lignin, but at the same time positively correlated with the cellulose degree of polymerization, weight-average molecular weight and hydrogen bond intensity. Four groups of compact clusters of hydrothermal pretreatment conditions were identified in a multivariate wood trait analysis.

Chalcone Synthase (CHS) Gene Suppression in Flax Leads to Changes in Wall Synthesis and Sensing Genes, Cell Wall Chemistry and Stem Morphology Parameters.

The chalcone synthase (CHS) gene controls the first step in the flavonoid biosynthesis. In flax, CHS down-regulation resulted in tannin accumulation and reduction in lignin synthesis, but plant growth was not affected. This suggests that lignin content and thus cell wall characteristics might be modulated through CHS activity. This study investigated the possibility that CHS affects cell wall sensing as well as polymer content and arrangement. CHS-suppressed and thus lignin-reduced plants showed significant changes in expression of genes involved in both synthesis of components and cell wall sensing. This was accompanied by increased levels of cellulose and hemicellulose. CHS-reduced flax also showed significant changes in morphology and arrangement of the cell wall. The stem tissue layers were enlarged averagely twofold compared to the control, and the number of fiber cells more than doubled. The stem morphology changes were accompanied by reduction of the crystallinity index of the cell wall. CHS silencing induces a signal transduction cascade that leads to modification of plant metabolism in a wide range and thus cell wall structure.

Time Course Field Analysis of COMT-Downregulated Switchgrass: Lignification, Recalcitrance, and Rust Susceptibility

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38 % more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2 years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11–16 % less lignin, 33–40 % lower S/G (syringyl-to-guaiacyl) ratios, and 15–42 % higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. These results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release.

SummaryPlant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand‐full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.

Bacteria-Assisted Synthesis of Electrode Materials for Li or Na Batteries

The production of materials for high-energy Li-ion batteries is generally associated with negative environmental footprints, due to high temperatures required and chemical toxicity of their components [1]. Our goal is to explore new bio-assisted low-temperature routes of synthesis in order to reduce this environmental impact and possibly improve their electrochemical performances. Bacteria are easy to grow; able to mineralize transition metals and other elements [2], with controlled morphology, crystal structure and texture; they also constitute a potential source of conductive carbon. For these reasons, bacteria assisted synthesis caught our attention, especially as exceptional power performances were already shown for biomineralized hollow shells of hematite (α-Fe2O3) [3] when compared to those of non-textured material. Hydrated amorphous ferric phosphate (a-FeIIIPO4·~3H2O) was precipitated from aqueous solution at 30°C under air at the contact of bacteria (Sporosarcina pasteurii). The resulting materials were investigated using various techniques (e.g. thermal analysis, mass spectrometry, infrared spectroscopy, electron microscopy) and consist of dead bacteria (2-3 µm x 0.5 µm) coated by a 70 nm thick layer of interconnected a-FeIIIPO4·~3H2O particles (40-80 nm), as presented by the following microscopy image. Surface chemistry of the bacterial cell wall plays a key role in the nucleation process thereby controlling particle size. After a mild temperature treatment, a 90 mAh/g capacity has been achieved versus Li (at 3.0 Volts mean voltage) with an excellent cyclability, constant coulombic efficiency (~100%) and interesting power performances. This approach allows overcoming the intrinsic poor conductivity of Fe phosphate without any use of harsh mixing treatments with conductive additive, or complex synthetic procedure. Bacteria are capable of many things and we are now applying this strategy for other materials and other metals like manganese. We will also address the positive attributes of these bio-assisted synthesis for application in Na-based batteries. [1] Samaras C., Meisterling K., Environmental Science & Technology. 42, 3170 (2008). [2] Dupraz S. et al., Chemical Geology. 265, 54 (2009). [3] Miot J. et al., Energy & Environmental Science. 7, 451 (2014). Figure 1

Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.)

Key messageA switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.In recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

Using 2D NMR spectroscopy to assess effects of UV radiation on cell wall chemistry during litter decomposition

Litter chemistry is one of the most studied controls on decomposition in terrestrial ecosystems. Solar radiation has been shown to increase litter decomposition rates in arid ecosystems through the process of photodegradation. However, it remains unclear how photodegradation affects litter chemistry, especially the abundance and composition of lignin, which is thought to play a key role in photodegradation. Using two-dimensional nuclear magnetic resonance (2D NMR) spectroscopic methods, we quantified the molecular-level changes in litter chemistry associated with photodegradation. Litter of Bromus diandrus was exposed in the field to two levels of radiation [with and without ultraviolet (UV) wavelengths] and two durations of exposure (2.5 months during summer, and 1 year). Through fiber analysis by sequential digestion, we found that the litter hemicellulose fraction decreased significantly from 31.6 to 24.9 % after 1 year of decomposition. In litter exposed for 1 year, the hemicellulose fraction was significantly lower in litter with UV exposure compared to litter without UV exposure (23.8 vs. 25.9 %). These results indicate that UV photodegradation has a small but significant effect on litter chemistry compared to other decomposition processes. Even though fiber analysis showed no loss of total lignin, 2D NMR analysis demonstrated that UV exposure reduced the major lignin structural units containing β-aryl ether inter-unit linkages by 9 % and decreased the relative abundance of lignin p-hydroxyphenyl units by 20 %. The 2D NMR analysis also revealed that lignin guaiacyl units were preferentially lost after 1 year of decomposition relative to the reference material, but no effects of UV exposure on guaiacyl units were observed. These results suggest that photodegradation causes partial degradation, not necessarily complete breakdown, of lignin structures. Our data also demonstrate that applications of 2D NMR methods are valuable for acquiring detailed information on lignin and polysaccharide chemistry during both biotic and abiotic decomposition processes.

Visualization of Miscanthus×giganteus cell wall deconstruction subjected to dilute acid pretreatment for enhanced enzymatic digestibility.

BackgroundThe natural recalcitrance of lignocellulosic plant cell walls resulting from complex arrangement and distribution of heterogeneous components impedes deconstruction of such cell walls. Dilute acid pretreatment (DAP) is an attractive method to overcome the recalcitrant barriers for rendering enzymatic conversion of polysaccharides. In this study, the internodes of Miscanthus × giganteus, a model bioenergy crop, were subjected to DAP to yield a range of samples with altered cell wall structure and chemistry. The consequent morphological and compositional changes and their possible impact on saccharification efficiency were comprehensively investigated. The use of a series of microscopic and microspectroscopic techniques including fluorescence microscopy (FM), transmission electron microscopy (TEM) and confocal Raman microscopy (CRM)) enabled correlative cell wall structural and chemical information to be obtained.ResultsDAP of M. × giganteus resulted in solubilization of arabinoxylan and cross-linking hydroxycinnamic acids in a temperature-dependent manner. The optimized pretreatment (1% H2SO4, 170°C for 30 min) resulted in significant enhancement in the saccharification efficiency (51.20%) of treated samples in 72 h, which amounted to 4.4-fold increase in sugar yield over untreated samples (11.80%). The remarkable improvement could be correlated to a sequence of changes occurring in plant cell walls due to their pretreatment-induced deconstruction, namely, loss in the matrix between neighboring cell walls, selective removal of hemicelluloses, redistribution of phenolic polymers and increased exposure of cellulose. The consequently occurred changes in inner cell wall structure including damaging, increase of porosity and loss of mechanical resistance were also found to enhance enzyme access to cellulose and further sugar yield.ConclusionsDAP is a highly effective process for improving bioconversion of cellulose to glucose by breaking down the rigidity and resistance of cell walls. The combination of the most relevant microscopic and microanalytical techniques employed in this work provided information crucial for evaluating the influence of anatomical and compositional changes on enhanced enzymatic digestibility.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0282-3) contains supplementary material, which is available to authorized users.