Mapping of Cell Wall Components in Lignified Biomass as a Tool to Understand Recalcitrance
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Keywords:
Hemicellulose
Secondary cell wall
Secondary cell walls (SCWs) are produced by specialized plant cell types, and are particularly important in those cells providing mechanical support or involved in water transport. As the main constituent of plant biomass, secondary cell walls are central to attempts to generate second-generation biofuels. Partly as a consequence of this renewed economic importance, excellent progress has been made in understanding how cell wall components are synthesized. SCWs are largely composed of three main polymers: cellulose, hemicellulose, and lignin. In this review, we will attempt to highlight the most recent progress in understanding the biosynthetic pathways for secondary cell wall components, how these pathways are regulated, and how this knowledge may be exploited to improve cell wall properties that facilitate breakdown without compromising plant growth and productivity. While knowledge of individual components in the pathway has improved dramatically, how they function together to make the final polymers and how these individual polymers are incorporated into the wall remain less well understood.
Secondary cell wall
Hemicellulose
Plant cell
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We have developed a novel 3-D, agent-based model of cell-wall digestion to improve our understanding of ruminal cell-wall digestion. It offers a capability to study cell walls and their enzymatic modification, by providing a representation of cellulose microfibrils and non-cellulosic polysaccharides and by simulating their spatial and catalytic interactions with enzymes. One can vary cell-wall composition and the types and numbers of enzyme molecules, allowing the model to be applied to a range of systems where cell walls are degraded and to the modification of cell walls by endogenous enzymes. As a proof of principle, we have modelled the wall of a mesophyll cell from the leaf of perennial ryegrass and then simulated its enzymatic degradation. This is a primary, non-lignified cell wall and the model includes cellulose, hemicelluloses (glucuronoarabinoxylans, 1,3;1,4-β-glucans, and xyloglucans) and pectin. These polymers are represented at the level of constituent monosaccharides, and assembled to form a 3-D, meso-scale representation of the molecular structure of the cell wall. The composition of the cell wall can be parameterised to represent different walls in different cell types and taxa. The model can contain arbitrary combinations of different enzymes. It simulates their random diffusion through the polymer networks taking collisions into account, allowing steric hindrance from cell-wall polymers to be modelled. Steric considerations are included when target bonds are encountered, and breakdown products resulting from enzymatic activity are predicted.
Pectin
Secondary cell wall
Xyloglucan
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Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.
Hemicellulose
Secondary cell wall
Digestion
Enzymatic Hydrolysis
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The goal of this project is to increase the fundamental understanding of the plant secondary cell wall in wood formation, using Populus trichocarpa as the model species. The project is built on a systematic transgenic strategy to perturb the expression of targeted cell-wall component genes and transcription factors (TFs). We used a systems biology approach that integrates this genome-wide transgenesis with full genome information to advance our understanding of wood formation and how to engineer woody plants to create robust feedstocks that maximize beneficial traits for biofuel and material production. This work has resulted in 14 peer reviewed publications in journals such as Plant Cell, Proceedings of the National Academy of Sciences, Nature Protocols, Journal of Proteome Research, Plant Biotechnology Journal, etc. and in 13 conference presentations. The project’s full genome RNA-seq data sets are available through Gene Expression Omnibus (GSE49911 and GSE81077). 7 PhD and 2 MS graduate students received degrees under full or partial support of this grant. We also supported 3 undergraduates, 5 post-docs and 3 visiting scientists. Total 30 RNAi and artificial microRNA (amiRNA) transgene constructs were prepared for knocking down the genes involved in cellulose and hemicellulose biosynthesis and constructs for overexpressing TFs that control wood formation. The project generated over 1,000 transgenic P. trichocarpa and made many novel contributions to the research field, as outline below. We established a simple ihigh-throughput xylem protoplast system for studying wood formation. The system can be used as a cellular model to study gene transactivation and nucleocytoplasmic protein trafficking, and is particularly useful for studies where stable transgenics and mutants are unavailable. Our system is markedly faster and provides better yields than previous protocols. We further developed the first chromatin immunoprecipitation (ChIP) procedures for P. trichocarpa and many other woody species. We used this protocol to identify genome-wide specific TF-DNA interactions and histone modifications associated with wood formation. Our protocol is suitable for many tissue types and is so far the only working ChIP system for wood-forming tissue. We discovered a stem-differentiating xylem (SDX)-specific alternative SND1 transcription factor (TF) splice variant, PtrSND1-A2IR, that acts as a dominant negative of SND1 transcriptional network genes in Populus trichocarpa. PtrSND1-A2IR is exclusively in cytoplasmic foci but translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. The translocated PtrSND1-A2IR can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. We further discovered another splice variant, PtrVND6-C1IR, derived from PtrVND6-C1. Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. However, these splice variants from the PtrVND6 and PtrSND1 families may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation. We next developed a mass spectrometry (MS) based absolute quantification of SDX proteins including TFs, which are normal present in very low concentration. With our digestion optimization, subcellular fractionation of SDX nuclei, and improved instrumentation in the quadrupole orbitrap MS, we were able to identify over 6,000 unique proteins, including cellulose and hemicellulose biosynthesis proteins as well as many SND and VND TF members. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa and used these transgenics built a hierarchical genetic regulatory network (GRN), which demonstrated that ptr-miR397a is a negative regulator of laccases for lignin biosynthesis. The GRN further identified previously undisclosed TFs and their targets including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. We also analyzed the transcriptomes of 5 tissues (xylem, phloem, shoot, leaf, and root) and 2 wood forming cell-types (fiber and vessel) of P. trichocarpa to assemble gene co-expression subnetworks. We identified 165 TFs that showed xylem-, fiber-, and vessel-specific expression. Of the 165 TFs, 101 co-expressed with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Our co-expression network suggests a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.
Hemicellulose
Secondary cell wall
Populus trichocarpa
Synthetic Biology
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Plants are comprised of different particular cell types that contrast in their cell wall arrangement and structure. The cell walls of specific tissues like xylem sclerenchyma are portrayed by the occurrence of cellulose and the heterogeneous lignin polymer all of which assumes a noteworthy role in the physiology plant growth and for the sustainable economic purposes like bioethanol production. By far most of plant biomass comprises of various cell wall polymers created by living plant cells. The greater part of these polymers are vitality rich connected sugars that shape the major auxiliary system in plant cell walls, especially in the thick secondary cell wall describing certain tissues. Notwithstanding cell wall polysaccharides, another critical cell wall biopolymer is lignin restricting the access to cell wall sugars Because of its huge financial effect and pivotal job in vascular plant advancement; lignification is an imperative topic in plant biochemistry. So it is really important to understand the intricate network of secondary cell wall components and their biosynthesis which will be the major highlights for discussion in this review.
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Hemicellulose
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Efficient conversion of lignocellulosic biomass to second-generation biofuels and valuable chemicals requires decomposition of resilient plant cell wall structure. Cell wall recalcitrance varies among plant species and even phenotypes, depending on the chemical composition of the noncellulosic matrix. Changing the amount and composition of branches attached to the hemicellulose backbone can significantly alter the cell wall strength and microstructure. We address the effect of hemicellulose composition on primary cell wall assembly forces by using the 3D-RISM-KH molecular theory of solvation, which provides statistical-mechanical sampling and molecular picture of hemicellulose arrangement around cellulose. We show that hemicellulose branches of arabinose, glucuronic acid, and especially glucuronate strengthen the primary cell wall by strongly coordinating to hydrogen bond donor sites on the cellulose surface. We reveal molecular forces maintaining the cell wall structure and provide directions for genetic modulation of plants and pretreatment design to render biomass more amenable to processing.
Hemicellulose
Glucuronates
Secondary cell wall
Lignocellulosic Biomass
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Plant cell
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Hemicellulose
Xyloglucan
Monosaccharide
Secondary cell wall
Pectin
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The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.
Miscanthus sinensis
Xyloglucan
Arabinoxylan
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