Focus Issue on Plant Cell Walls: A Comprehensive Toolkit of Plant Cell Wall Glycan-Directed Monoclonal Antibodies

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.Cell walls play important roles in the structure, physiology, growth, and development of plants (Carpita and Gibeaut, 1993). Plant cell wall materials are also important sources of human and animal nutrition, natural textile fibers, paper and wood products, and raw materials for biofuel production (Somerville, 2007). Many genes thought to be responsible for plant wall biosynthesis and modification have been identified (Burton et al., 2005; Lerouxel et al., 2006; Mohnen et al., 2008), and 15% of the Arabidopsis (Arabidopsis thaliana) genome is likely devoted to these functions (Carpita et al., 2001). However, phenotypic analysis in plants carrying cell wall-related mutations has proven particularly difficult. First, cell wall-related genes are often expressed differentially and at low levels between cells of different tissues (Sarria et al., 2001). Also, plants have compensatory mechanisms to maintain wall function in the absence of a particular gene (Somerville et al., 2004). Thus, novel tools and approaches are needed to characterize wall structures and the genes responsible for their synthesis and modification.Monoclonal antibodies (mAbs) developed against cell wall polymers have emerged as an important tool for the study of plant cell wall structure and function (Knox, 2008). Previous studies have utilized mAbs that bind epitopes present on rhamnogalacturonan I (RG-I; Freshour et al., 1996; Jones et al., 1997; Willats et al., 1998; McCartney et al., 2000; Clausen et al., 2004; Altaner et al., 2007), homogalacturonan (Willats et al., 2001; Clausen et al., 2003), xylogalacturonan (Willats et al., 2004), xylans and arabinoxylans (McCartney et al., 2005), xyloglucan (Freshour et al., 1996, 2003; Marcus et al., 2008), arabinogalactan(protein) (Pennell et al., 1991; Puhlmann et al., 1994; Dolan et al., 1995; Smallwood et al., 1996), and extensins (Smallwood et al., 1995) to localize these epitopes in plant cells and tissues. In addition, mAbs have been used to characterize plants carrying mutations in genes thought to be associated with cell wall biosynthesis and metabolism (Orfila et al., 2001; Seifert, 2004; Persson et al., 2007; Cavalier et al., 2008; Zabotina et al., 2008). Despite their utility, the available set of mAbs against carbohydrate structures is relatively small given the structural complexity of wall polymers (Ridley et al., 2001; O''Neill and York, 2003), and knowledge of their epitope specificity is limited. Thus, additional mAbs specific to diverse epitope structures and methods for rapid epitope characterization are needed (Somerville et al., 2004).Here, we report the generation of 130 new mAbs that bind to diverse epitopes present on a broad spectrum of plant cell wall glycans. In addition, approximately 50 previously reported or generated mAbs were included in the ELISA-based screens used to group the antibodies according to their binding patterns against a diverse panel of 54 polysaccharides. The resulting ELISA data were analyzed by hierarchical clustering to illustrate the relationships between the available mAbs. Nineteen groups of mAbs were identified from the clustering analysis. Some initial information regarding possible epitopes recognized by some of these antibodies could be inferred from the clustering analysis.     

Focus Issue on Plant Cell Walls: The Arabidopsis Family GT43 Glycosyltransferases Form Two Functionally Nonredundant Groups Essential for the Elongation of Glucuronoxylan Backbone

There exist four members of family GT43 glycosyltransferases in the Arabidopsis (Arabidopsis thaliana) genome, and mutations of two of them, IRX9 and IRX14, have previously been shown to cause a defect in glucuronoxylan (GX) biosynthesis. However, it is currently unknown whether IRX9 and IRX14 perform the same biochemical function and whether the other two GT43 members are also involved in GX biosynthesis. In this report, we performed comprehensive genetic analysis of the functional roles of the four Arabidopsis GT43 members in GX biosynthesis. The I9H (IRX9 homolog) and I14H (IRX14 homolog) genes were shown to be specifically expressed in cells undergoing secondary wall thickening, and their encoded proteins were targeted to the Golgi, where GX is synthesized. Overexpression of I9H but not IRX14 or I14H rescued the GX defects conferred by the irx9 mutation, whereas overexpression of I14H but not IRX9 or I9H complemented the GX defects caused by the irx14 mutation. Double mutant analyses revealed that I9H functioned redundantly with IRX9 and that I14H was redundant with IRX14 in their functions. In addition, double mutations of IRX9 and IRX14 were shown to cause a loss of secondary wall thickening in fibers and a much more severe reduction in GX amount than their single mutants. Together, these results provide genetic evidence demonstrating that all four Arabidopsis GT43 members are involved in GX biosynthesis and suggest that they form two functionally nonredundant groups essential for the normal elongation of GX backbone.Secondary walls constitute the bulk of cellulosic biomass produced by vascular plants. Cellulosic biomass in the form of fibers and wood is an important raw material for a myriad of industrial uses, such as timber, pulping, papermaking, and textiles. Due to the dwindling of nonrenewable fossil fuels and the detrimental effects of burning fossil fuels on the global environment, there has been an urgent call to develop alternative renewable energy sources, and the lignocellulosic biomass from plants is considered to be an attractive renewable source for biofuel production (Somerville, 2006). However, lignocellulosic biomass is recalcitrant to the enzymatic conversion of cellulose into sugars, because cellulose is embedded in a complex mixture of polysaccharides and lignin polymers that block the accessibility of degrading enzymes. It has been shown that reduction of lignin and xylan by chemical or enzymatic treatment or by the transgenic approach reduces the recalcitrance of the lignocellulosic biomass to saccharification (Chen and Dixon, 2007; Himmel et al., 2007; Lee et al., 2009a). Therefore, a complete understanding of how individual components of lignocellulosic biomass are biosynthesized will potentially allow us to design novel strategies for genetic modification of cell wall composition and, hence, reduction in biomass recalcitrance to biofuel production.Xylan is the main hemicellulose that cross-links with cellulose in the secondary walls of dicot plants (Carpita and McCann, 2000). It is made of a linear backbone of β-(1,4)-linked xylosyl residues, about 10% of which are attached with side chains of single residues of glucuronic acid (GlcA) and/or 4-O-methylglucuronic acid (MeGlcA) via α-(1,2)-linkages. The backbone xylosyl residues may also be substituted with the arabinosyl group and acetylated. Based on the nature of the side chains, xylan is generally grouped as (methyl)glucuronoxylan (GX), which is the main hemicellulose in dicots, and arabinoxylan and glucuronoarabinoxylan, which are the most abundant hemicelluloses in grass cell walls (Ebringerová and Heinze, 2000). In addition to the xylosyl backbone, the reducing end of xylan from birch (Betula verrucosa), spruce (Picea abies), Arabidopsis (Arabidopsis thaliana), and poplar (Populus alba × Populus tremula) contains a unique tetrasaccharide sequence β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (Shimizu et al., 1976; Johansson and Samuelson, 1977; Andersson et al., 1983; Peña et al., 2007; Lee et al., 2009a).The biosynthesis of xylan requires multiple glycosyltransferases and other modifying enzymes. Early biochemical studies revealed the activities of xylosyltransferases, glucuronosyltransferases, arabinosyltransferases, methyltransferases, and acetyltransferases that are likely involved in the biosynthesis of xylan (Baydoun et al., 1983, 1989; Kuroyama and Tsumuraya, 2001; Gregory et al., 2002; Porchia et al., 2002; Urahara et al., 2004; Zeng et al., 2008). However, none of the genes corresponding to these xylan biosynthetic enzymes have been identified. Recent molecular and genetic studies in Arabidopsis and poplar have led to the identification of a number of glycosyltransferases that are essential for GX biosynthesis. Among them, several members of the families GT47 and GT8 from Arabidopsis (FRA8, F8H, IRX8, and PARVUS) and poplar (GT47C, GT8D, and GT8E/8F) are implicated in the biosynthesis of the GX reducing end sequence (Aspeborg et al., 2005; Brown et al., 2005, 2007; Zhong et al., 2005; Zhou et al., 2006, 2007; Lee et al., 2007b, 2009b, 2009c; Peña et al., 2007; Persson et al., 2007). These glycosyltransferase genes are specifically expressed in vessels and fibers, and their encoded proteins are targeted to Golgi, where GX is synthesized, except for PARVUS and GT8E/8F, which are predominantly located in the endoplasmic reticulum (Lee et al., 2007b, 2009c). Mutations of the Arabidopsis FRA8, IRX8, and PARVUS genes all led to a near loss of the reducing end tetrasaccharide sequence and a reduction in GX amount (Brown et al., 2007; Lee et al., 2007b; Peña et al., 2007), indicating their essential roles in the biosynthesis of the GX reducing end sequence, although their exact enzymatic activities are still unknown.The genetic studies have also identified roles of two members of family GT43 glycosyltransferases, IRX9 and IRX14, from Arabidopsis and GT43B from poplar in the biosynthesis of the GX xylosyl backbone (Brown et al., 2007; Peña et al., 2007; Zhou et al., 2007). The expression of IRX9 has been shown to be associated with cells undergoing secondary wall biosynthesis, and its encoded protein is targeted to the Golgi. Mutation of the IRX9 gene causes a drastic reduction in xylan xylosyltransferase activity (Brown et al., 2007; Lee et al., 2007a) and concomitantly a substantial decrease in the GX chain length and GX amount (Peña et al., 2007). Mutation of IRX14 was shown to result in a reduction in the GX level and the xylosyltransferase activity (Brown et al., 2007). In addition, two functionally redundant glycosyltransferases, IRX10 and IRX10-like, which belong to family GT47, were also demonstrated to be required for the normal GX level and xylan xylosyltransferase activity, suggesting their involvement in the biosynthesis of the GX xylosyl backbone (Brown et al., 2009; Wu et al., 2009).In this report, we performed comprehensive molecular and genetic studies of the roles of all members of the Arabidopsis family GT43 glycosyltransferases in GX biosynthesis. We show that, like IRX9, the other three GT43 members, I9H (IRX9 homolog), IRX14, and I14H (IRX14 homolog), are expressed in secondary wall-containing cells and that their encoded proteins are targeted to the Golgi. We have found that the GX defects in the irx9 mutant can be rescued by overexpression of I9H but not IRX14 and I14H. Similarly, overexpression of I14H but not IRX9 and I9H is able to complement the GX defects caused by the irx14 mutation. Furthermore, genetic analysis of an array of double mutants revealed redundant and nonredundant roles of GT43 members in GX biosynthesis. Our findings demonstrate that the Arabidopsis family GT43 glycosyltransferases form two functionally nonredundant groups essential for the normal elongation of GX backbone.     

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.     

Celebrating Plant Cells： A Special Issue on Plant Cell Biology

A special issue on plant cell biology is long overdue for JIPB! In the last two decades or so, the plant biology community has been thrilled by explosive discoveries regarding the molecular and genetic basis of plant growth, development, and responses to the environment, largely owing to recent maturation of model systems like Arabidopsis thaliana and the rice Oryza sativa, as well as the rapid development of high throughput technologies associated with qenomics and proteomics.     

Equilibrium Analysis of Calcium Binding to Cell Walls Isolated from Plant Tissue

Primary cell walls were isolated and purified from potato tubersand carrots via a Parr N₂ bomb technique. Calcium binding topurified cell walls was measured with both calcium selectiveelectrode and use of the metallochromic indicator, ArsenazoIII. The cell walls used in this study were biologically activeand presumably approached the physiological cell wall. Aliquotsof the untreated cell walls (control) were then salt-extractedor EDTA-treated and binding properties were compared to thecontrols. In addition, the binding properties of freshly preparedcell walls were compared to cell walls which were stored for1 week at 2°C. Both simple Scatchard plot analysis and anelectrostatic interaction model were used to evaluate calciumbinding parameters. The controls from the two tissue types hadinherently different calcium binding properties and these propertieswere affected by treating the cell walls with salt or EDTA.Cold storage treatment drastically changed the binding propertiesof carrot cell walls but had negligible effect on potato tubercell walls. (Received January 28, 1992; Accepted April 3, 1992)     

The Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension-cultured Monocots

The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed β-1,3; β-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included.     

Structure of Plant Cell Walls: VIII. A New Pectic Polysaccharide

This paper describes the isolation and characterization of rhamnogalacturonan II, a hitherto unobserved component of the primary cell walls of dicotyledonous plants. Rhamnogalacturonan II constitutes 3 to 4% of the primary cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells. Rhamnogalacturonan II is a very complex polysaccharide yielding, upon hydrolysis, 10 different monosaccharides including the rarely observed sugars apiose, 2-O-methylxylose, and 2-O-methylfucose. In addition, rhamnogalacturonan II is characterized by the rarely observed glycosyl interconnections of 2-linked glucuronosyl, 3,4-linked fucosyl, and 3-linked rhamnosyl residues. These glycosyl linkages have never previously been detected in primary sycamore cell walls. Evidence is presented which suggests that polysaccharides similar to rhamnogalacturonan II are present in the primary cell walls of the three other dicotyledonous plants examined.     

Focus Issue on Roots: Plant Nutrition: Root Transporters on the Move

Nutrient and water uptake from the soil is essential for plant growth and development. In the root, absorption and radial transport of nutrients and water toward the vascular tissues is achieved by a battery of specialized transporters and channels. Modulating the amount and the localization of these membrane transport proteins appears as a way to drive their activity and is essential to maintain nutrient homeostasis in plants. This control first involves the delivery of newly synthesized proteins to the plasma membrane by establishing check points along the secretory pathway, especially during the export from the endoplasmic reticulum. Plasma membrane-localized transport proteins are internalized through endocytosis followed by recycling to the cell surface or targeting to the vacuole for degradation, hence constituting another layer of control. These intricate mechanisms are often regulated by nutrient availability, stresses, and endogenous cues, allowing plants to rapidly adjust to their environment and adapt their development.Plants take up nutrients and water from the soil and transport them to the leaves to support photosynthesis and plant growth. However, most soils around the world do not provide optimal conditions for plant colonization. Consequently, plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients and water supply. Membrane transport proteins, including channels and transporters, play crucial roles in the uptake of nutrients and water from the soil and in their radial transport to the root vasculature. Newly synthesized membrane transport proteins have to be properly targeted to a defined compartment, usually the plasma membrane, to efficiently ensure their function. The trafficking of membrane transport proteins along the secretory pathway is tightly controlled and involves the recognition of exit signals by gatekeeper protein complexes. After reaching the plasma membrane, membrane transport proteins can be endocytosed and subsequently recycled to the cell surface or targeted to the vacuole for degradation. Because the subcellular localization of proteins directly influences their activity, modulating the localization of membrane transport proteins constitutes a powerful way to control nutrient and water uptake in plants. This review discusses the fundamental mechanisms at stake in membrane protein secretion and endocytosis, with a specific focus on membrane transport proteins, and how endogenous and exogenous cues affect their dynamics to integrate uptake of nutrients and water to plant growth conditions.     

Focus Issue on Roots: Perennial Roots to Immortality,

Maximum lifespan greatly varies among species, and it is not strictly determined; it can change with species evolution. Clonal growth is a major factor governing maximum lifespan. In the plant kingdom, the maximum lifespans described for clonal and nonclonal plants vary by an order of magnitude, with 43,600 and 5,062 years for Lomatia tasmanica and Pinus longaeva, respectively. Nonclonal perennial plants (those plants exclusively using sexual reproduction) also present a huge diversity in maximum lifespans (from a few to thousands of years) and even more interestingly, contrasting differences in aging patterns. Some plants show a clear physiological deterioration with aging, whereas others do not. Indeed, some plants can even improve their physiological performance as they age (a phenomenon called negative senescence). This diversity in aging patterns responds to species-specific life history traits and mechanisms evolved by each species to adapt to its habitat. Particularities of roots in perennial plants, such as meristem indeterminacy, modular growth, stress resistance, and patterns of senescence, are crucial in establishing perenniality and understanding adaptation of perennial plants to their habitats. Here, the key role of roots for perennial plant longevity will be discussed, taking into account current knowledge and highlighting additional aspects that still require investigation.There is enormous diversity among the types of perennial plants and among their patterns of aging (Jones et al., 2014). Perennial plants can be divided into herbaceous (or perennial herbs) and woody perennials (trees and shrubs), and therefore, they represent very diverse organisms in size and complexity from some herbs that weigh a few grams to huge trees like sequoias (Sequoia sempervirens). Among perennial herbs, the slowest growing species described thus far, Borderea pyrenaica (a small geophyte growing in the Central Pyrenees of northeastern Spain), is also the one with the longest maximum lifespan (350 years; Fig. 1). Interestingly, fecundity of this species increases with aging, representing a case of negative senescence (Garcia et al., 2011; Morales et al., 2013). If mortality falls as size increases and if size increases with age, then mortality will fall with age, and negative senescence occurs (Vaupel et al., 2004). Negative senescence is not common in the tree of life, but it seems to occur in not only some perennial herbs, such as B. pyrenaica (Garcia et al., 2011) and Plantago lanceolata (Roach and Gampe, 2004), but also, other phylogenetically distant organisms, such as turtles (Jones et al., 2014). Other perennial herbs with higher biomass production rates and consequently, larger sizes, such as stinging nettle (Urtica dioica), are much shorter-lived (a few years only). In this case, however, perenniality is achieved by allocating an important part of their energy to asexual reproduction (production of stolons; i.e. clonal propagation), giving rise to new entire clonal plants (Koskela, 2002). Indeed, this process happens in several other plant species with rapid growth that we commonly find in gardens, such as strawberries (Fragaria × ananassa) or raspberries (Rubus idaeus). Stolons can be produced aboveground or underground (in the latter case, forming rhizomes). Van Dijk (2009) elegantly reviewed the direct and indirect methods currently used to estimate plant age in clonal and nonclonal plants, showing several examples of plant species using clonal propagation with maximum lifespans of thousands of years, with the most notable example, King’s Lomatia (Lomatia tasmanica), being dated at 43,600 years (Lynch et al., 1998). Only one wild-living clone of this species is known. Clonal propagation is the only means for propagation, because it is a sterile ancient clone. When a branch falls, that branch produces new roots, establishing a new plant that is genetically identical to its parent (Lynch et al., 1998). Here, the production of new roots becomes essential for achieving potential immortality. Another example of extreme longevity is the bristlecone pine (Pinus longaeva), with a maximum lifespan of 5,062 years. It holds the record of longevity of a single individual within the plant kingdom, which was observed by Tom Harlan during 2012 in a living individual of this species in the White Mountains (the location has not been reported; Earle, 2013).Open in a separate windowFigure 1.Examples of extreme longevity in perennial plants. A, B. pyrenaica, the perennial herb with the longest lifespan described to date. B, A cross section of the tuber of B. pyrenaica showing the scars left by the five meristematic points in the spiral. C, P. longaeva, the species with the individual with the longest lifespan ever recorded (not using clonal propagation). D, C. nodosa meadow, with a detail of the rhizomes (E) that allow clonal propagation and potential immortality in this species. [See online article for color version of this figure.]The enormous diversity in lifespans within a species responds to specific life history traits and mechanisms evolved by each individual to adapt to its habitat. Particularities of roots in perennial plants, such as meristem indeterminacy, modular growth, stress resistance, and patterns of senescence, are crucial in understanding adaptation of perennial plants to their habitats, explaining differences in longevity. Here, the key role of roots in providing long lifespans in perennial plants will be discussed, taking into account current knowledge and highlighting additional aspects that still require investigation.     

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation¹ and other products such as biocomposite materials⁷. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin⁶ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization³, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units⁵. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II².