Illuminating the wall: Using click chemistry to image pectins in Arabidopsis cell walls

Plant cell walls are the most abundant biomaterials on Earth and serve a multitude of purposes in human society. These complex extracellular matrices are mainly composed of polysaccharides, including cellulose, hemicelluloses, and pectins, which cannot be cytologically examined using conventional techniques. Click chemistry, which exploits a bio-orthogonal cycloaddition reaction between alkynyl and azido groups, has proven to be useful for the metabolic incorporation and detection of modified sugars in polysaccharides in animals, fungi, and bacteria, but its use to interrogate the biosynthesis or dynamics of plant cell walls has not been previously reported. Recently, we found that an alkynylated analog of fucose can be metabolically incorporated into Arabidopsis thaliana cell walls and click labeled with fluorescent probes, facilitating imaging of cell wall carbohydrates. Despite the presence of fucose in several classes of wall polysaccharides, fucose-alkyne was primarily incorporated into rhamnogalacturonan-I, a type of pectin. Using timecourse and pulse-labeling experiments, we observed the dynamics of pectin delivery and reorganization in expanding cell walls. The use of click chemistry to investigate plant cell wall architecture should help bridge the gap between biochemical characterization of isolated cell wall components and an understanding of how those components interact in intact cell walls.     

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.     

Plant cell wall characterization using scanning probe microscopy techniques

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy.     

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.     

植物果胶的生物合成与功能

果胶作为植物细胞壁多糖之一,其结构和功能非常复杂。果胶主要由同型半乳糖醛酸聚糖(HG)、鼠李半乳糖醛酸聚糖I (RGI)和鼠李半乳糖醛酸聚糖II (RGII)组成。果胶类成分在维持细胞壁结构的完整性以及细胞间黏附和信号转导等方面发挥重要作用。研究果胶类成分的结构、分布和功能,对理解细胞壁高级结构的构建和功能具有重要意义...     

Primary Cell Wall Structure in the Evolution of Land Plants

Investigation of the primary cell walls of lower plants improves our understanding of the cell biology of these organisms but also has the potential to improve our understanding of cell wall structure and function in angiosperms that evolved from lower plants. Cell walls were prepared from eight species, ranging from a moss to advanced gymnosperms, and subjected to sequential chemical extraction to separate the main polysaccharide fractions. The glycosyl compositions of these fractions were then determined by gas chromatography. The results were compared among the eight plants and among data from related studies reported in the existing published reports to identify structural features that have been either highly conserved or clearly modified during evolution. Among the highly conserved features are the presence of a cellulose framework, the presence of certain hemicelluloses such as xyloglucan, and the presence of rhamnogalacturonan II, a domain in pectic polysaccharides. Among the modified features are the abundance of mannosyl-containing hemicelluloses and the presence of methylated sugars.     

Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era

Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified. This review focuses on the synthesis of non-cellulosic matrix polysaccharides formed in the Golgi apparatus. Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases. Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene. This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes. However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes. Database searches using various strategies and expression profiling can identify candidate genes. The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods. Recent advances using these methods are considered in this review.     

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.     

The plant cell wall: Biosynthesis,construction, and functions

The plant cell wall is composed of multiple biopolymers, representing one of the most complex structural networks in nature. Hundreds of genes are involved in building such a natural masterpiece. However, the plant cell wall is the least understood cellular structure in plants. Due to great progress in plant functional genomics,manyachievementshavebeenmadein uncovering cell wall biosynthesis, assembly, and architecture, as well as cell wall regulation and signaling. Such information has significantly advanced our understanding of the roles of the cell wall in many biological and physiological processes and has enhanced our utilization of cell wall materials. The use of cutting-edge technologies such as single-molecule imaging,nuclear magnetic resonance spectroscopy, and atomic force microscopy has provided much insight into the plant cell wall as an intricate nanoscale network, opening up unprecedented possibilities for cell wall research. In this review,we summarize the major advances made in understanding the cell wall in this era of functional genomics, including the latest findings on the biosynthesis, construction, and functions of the cell wall.     

Pectin structure and biosynthesis

Pectin is structurally and functionally the most complex polysaccharide in plant cell walls. Pectin has functions in plant growth, morphology, development, and plant defense and also serves as a gelling and stabilizing polymer in diverse food and specialty products and has positive effects on human health and multiple biomedical uses. Pectin is a family of galacturonic acid-rich polysaccharides including homogalacturonan, rhamnogalacturonan I, and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA). Pectin biosynthesis is estimated to require at least 67 transferases including glycosyl-, methyl-, and acetyltransferases. New developments in understanding pectin structure, function, and biosynthesis indicate that these polysaccharides have roles in both primary and secondary cell walls. Manipulation of pectin synthesis is expected to impact diverse plant agronomical properties including plant biomass characteristics important for biofuel production.     

QUASIMODO 3 (QUA3) is a putative homogalacturonan methyltransferase regulating cell wall biosynthesis in Arabidopsis suspension-cultured cells

Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.     

Rice cell wall polysaccharides: Structure and biosynthesis

Rice is the most widely consumed staple food, and is cultivated worldwide to satisfy our daily caloric needs. Thus, extensive efforts on rice breeding and biotechnology have substantially focused on the development of elite cultivars with high yields and better grain quality, as well as enhanced resistance against biotic and abiotic stresses. Recently, it has been observed that rice is more than a just grain-producing crop. Carbon-rich materials of the rice cell wall polysaccharides from post-harvest wastes, including the straw and husk, have been converted into bioethanol and other invaluable, renewable materials. In order to maximize the utilization of cell wall-derived resources, it is imperative to understand cell wall chemistry and molecular mechanism underlying cell wall biosynthesis in rice. In the last decade, several approaches, including mutational genetics and the functional characterization of candidate genes, have been successful in isolating some of cell wall biosynthetic genes in rice, marking the first step forward in obtaining a complete understanding of rice cell wall biosynthesis, although the exact biochemical functions have not been conclusive. In this paper, we focus on integrating old and new information to provide an updated perspective in the cell wall formation of rice, highlighting the chemical structures and biosynthesis of rice cell wall polysaccharides.     

Determining the polysaccharide composition of plant cell walls

The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.     

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging

Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin, whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix. Understanding the distribution of these components and their functions within the cell wall can provide useful information on the biosynthesis of trees. Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose, as well as the functional groups of lignin in wood. In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution between these samples, while clear distinction was observed in the distribution of coniferyl alcohols and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood species control biosynthesis of lignin differently during the differentiation of cell wall.     

Polysaccharidmetabolismus in den Zellwänden wachsender Keimlinge von Phaseolus aureus

Summary Quantitative determinations of the cell wall constituents (pectin, hemicellulose and -cellulose) of growing Phaseolus aureus seedlings showed marked changes during early growth. The cell walls of the 2 to 4 days old seedlings were composed of approximately 30% -cellulose, 50% hemicelluloses and 20% pectin. After four weeks the proportion of the different fractions had changed to approximately 60% -cellulose, 30% hemicelluloses and 10% pectin. Quantitative sugar determinations on these polysaccharide fractions have shown that mainly the non-cellulosic fractions (hemicelluloses and pectin) underwent considerable changes in sugar composition during growth. The hemicelluloses contained non-cellulosic polysaccharides with a high glucose content, which were not starch. These were broken down in the cell walls during growth.In a series of experiments in which ¹⁴C-glucose was injected into the hypocotyls of four days old Phaseolus aureus seedlings, the transport of radioactivity to the different plant organs and its incorporation into the cell wall polysaccharides of the bean stem were studied. The major part of the radioactivity was incorporated into the cell wall of the stem tissue. Minor amounts were transported to the roots and leaves. Of the cell wall polysaccharides of the stem, the hemicellulosic fraction showed a higher rate of incorporation of the ¹⁴C-glucose than the -cellulose in the early stages of growth. With increasing age of the plant, radioactivity was transferred from the hemicellulosic fraction to the -cellulose, suggesting turnover of polysaccharides in the growing cell wall.     

Changing extracellular matrix of cells during development of suspension culture of Triticum timopheevii Zhuk

A study was made of the contents of the main polysaccharide fractions in the cell wall, and extracellular polysaccharides, and of the activity of cell wall enzymes during cultivation of suspension culture of cells of the winter wheat Triticum timopheevii Zhuk. It was shown that within 3 days of cultivation (a phase enriched in dividing cells), on the background of increased callose contents in plant cells, amounts of pectins and hemicelluloses extracted by 4N alkali decreased. The content of polysaccharides reached its initial level by the end of culturing. A parallel analysis of glycosidase activity in cell walls has shown their considerable activation at the stage enriched by dividing cells, which decreased at a transition of culture into the stationary level. The increased activity of hydrolyzing enzymes was combined with an increased efflux of extracellular polysaccharides into culture medium. The detected changes in polysaccharide composition of the cell wall at the first phase indicate its qualitative changes during cell wall reconstruction at the beginning of cytokines, whereas extensive expansion of cell wall was seen on the phase of elongation.     

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Pectin is a family of complex polysaccharides present in all plant primary cell walls. The complicated structure of the pectic polysaccharides, and the retention by plants of the large number of genes required to synthesize pectin, suggests that pectins have multiple functions in plant growth and development. In this review we summarize the current level of understanding of pectin primary and tertiary structure, and describe new methods that may be useful to study localized pectin structure in the plant cell wall. We also discuss progress in our understanding of how pectin is biosynthesized and review the biological activities and possible modes of action of pectic oligosaccharides referred to as oligogalacturonides. We present our view of critical questions regarding pectin structure, biosynthesis, and function that need to be addressed in the coming decade. As the plant community works towards understanding the functions of the tens of thousands of genes expressed by plants, a large number of those genes are likely to be involved in the synthesis, turnover, biological activity, and restructuring of pectin. A combination of genetic, molecular, biochemical and chemical approaches will be necessary to fully understand the function and biosynthesis of pectin.     

细胞壁在植物重金属耐性中的作用

植物细胞壁主要是由多糖、蛋白质和木质素等组成的一个复合体,广泛参与植物生长发育及对各种逆境胁迫的响应,是重金属离子进入细胞质的第一道屏障。本文主要综述了植物细胞壁主要成分,包括细胞壁多糖、细胞壁蛋白质和木质素,在响应重金属胁迫反应中的作用及其参与重金属耐性的机制,以期能对植物细胞壁在重金属耐性中的作用有更深入的了解。