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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022. doi: 10.1101/glycobiology.4e.24
Viridiplantae (green plants) are a clade of photosynthetic organisms that contain chlorophylls a and b, produce and store their photosynthetic products inside a double-membrane-bounded chloroplast, and have cell walls that typically contain cellulose. As photoautotrophic organisms, green plants are capable of converting carbon dioxide to carbohydrates. Thus, carbohydrates are not limiting and their utilization throughout a plant's life cycle has expanded enormously both in functionality and structural diversity.
The Viridiplantae comprise two clades—the Chlorophyta and the Streptophyta. The Chlorophyta contain most of the organisms typically referred to as “green algae.” The term “algae” is also used for several other groups of photosynthetic eukaryotes, including diatoms and the red, brown, golden, and yellow-green algae. The Streptophyta comprise several other lineages that are also referred to as “green algae” and the land plants. Land plants include the liverworts, mosses, hornworts, lycopods, ferns, gymnosperms, and flowering plants. In this chapter, we provide an overview of the current knowledge of green plant glycan structures with an emphasis on the features that are unique to land plants.
Green plants synthesize diverse glycans and glycoconjugates that vary in structural complexity and molecular size. Soluble low-molecular-weight compounds encompass the carbon and energy transport disaccharide sucrose as well as glycoconjugates that contain aromatic (e.g., phenolic glycosides) or aliphatic (glycolipids) aglycones. Many of these compounds function in plant protection or defense (e.g., to ward off herbivores). Plant polysaccharides are linear or branched polymers composed of the same or different monosaccharides. Examples of homopolymers made entirely out of glucose include the storage polymer starch and the structural polymer cellulose (Figure 24.1). An example of a structurally complex polysaccharide is the plant cell wall pectic polysaccharide, referred to as rhamnogalacturonan II (RG-II), which contains 12 different monosaccharides linked together by up to 21 distinct glycosidic linkages (Figure 24.2). Plant proteoglycans are structurally diverse glycoconjugates in which carbohydrate, generally O-linked to the protein via hydroxyamino acids, accounts for up to 90% of the molecule (Figure 24.3). Plant glycoproteins typically contain 15% or less carbohydrate in the form of N-linked oligomannose, complex, hybrid, and paucimannose oligosaccharides (see Figure 24.4). Land plants also form O-GlcNAc-modified nuclear and cytosolic proteins (Chapter 19).
Glycosyl sequences of (A) cellulose, (B–E) selected hemicelluloses, (F) mixed-linkage glucan, and (G) callose.
Schematic structure of pectin. The three main pectic polysaccharides are shown: (RG-I) rhamnogalacturonan I; (HG) homogalacturonan; and (RG-II) rhamnogalacturonan II. The borate ester is formed between the apiosyl residue in side chain A of each RG-II (more...)
Schematic structure of the proteoglycan referred to as arabinoxylan pectin arabinogalactan protein1 (APAP1).
Types of N-glycans identified in plants.
Nucleotide sugars are the donors used for the synthesis of glycans, glycoconjugates, and glycosylated secondary metabolites (Chapter 5). In plants, the majority of these activated monosaccharides exist as their nucleotide-diphosphates (e.g., UDP-Glcp or GDP-Manp), although at least one monosaccharide, Kdo, exists as its cytidine monophosphate derivative (CMP-Kdop). Nucleotide sugars are formed from the carbohydrate generated by photosynthesis, from the monosaccharides released by hydrolysis of sucrose and storage carbohydrates, and by recycling monosaccharides from glycans and the cell wall. Nucleotide sugars are also formed by interconverting preexisting activated monosaccharides. To date, 30 different nucleotide sugars and at least 100 genes encoding proteins involved in their formation and interconversion have been identified in plants.
As plants are carbohydrate-rich organisms, it is perhaps not surprising that their genomes contain a large number of genes encoding proteins involved in the synthesis, metabolism, and modification of glycans and glycoconjugates. These proteins are spread across many enzyme classes in the Carbohydrate-Active enZYmes (CAZy) database (Table 24.1). Many of these proteins may be involved in the formation and modification of the polysaccharide-rich cell wall. Indeed, the unicellular alga Ostreococcus tauri, which is one of the few plants that does not form a cell wall, has a much smaller number of genes predicted to be involved in glycan metabolism.
Estimated number of genes encoding proteins involved in the synthesis and modification of glycans in plants and humans
The availability of plant lines carrying mutations in specific genes has yielded considerable insight into glycan biosynthesis and function. Arabidopsis thaliana has been widely used as a model dicot as it is easy to grow, has a short life cycle, and its relatively small genome has been sequenced and extensively annotated leading to the generation of chemically induced or transfer DNA (T-DNA) insertion mutant collections. These mutants have been vital in studying both the function and substrate specificity of diverse proteins involved in glycan synthesis and for demonstrating the function or redundancy of glycans and/or glycan substituents in the life-cycle of a plant. With the advent of low-cost whole-genome sequencing, the genomes of nearly 100 plant species including rice, maize, barley, poplar, and potato have been assembled (PlantGDB https://www.plantgdb.org). A large amount of transcriptomic data covering most green plant lineages is also available (https://sites.google.com/a/ualberta.ca/onekp/). With this sequence and annotation information, combined with gene editing techniques such as CRISPR/Cas9, plant mutants can be generated to test the functionality of genes related to glycans uniquely present in those plant species.
A forward genetic approach, in which Arabidopsis seeds are randomly chemically mutagenized and the resulting plants screened for structural changes in their glycans, has led to the discovery of multiple genes involved in nucleotide sugar interconversion pathways (Chapter 5) as well as plant cell wall–related glycosyltransferases (GTs) (Chapter 6). Reverse genetic approaches, with plants carrying loss-of-function mutations in known genes, have enabled the discovery of GTs involved in the synthesis of primary and secondary cell wall heteroxylans, pectins, N- and O-linked proteoglycans, glycolipids, and glycosylated metabolites. In addition, glycan-modifying enzymes including O-acetyl- and methyltransferases have been identified and characterized.
The initial product of photosynthesis, a triose phosphate, is used by plants in multiple ways. Triose phosphate can be converted to sucrose, a disaccharide of glucose and fructose (α-D-Glcp-1-2-β-D-Fruf) that is the most abundant soluble carbohydrate in green plants. Sucrose is the dominant transport carbohydrate for distributing the energy obtained by photosynthesis throughout the plant, in particular to nonphotosynthetic organs such as roots. Other water-soluble carbohydrates that are almost ubiquitous in the plant kingdom include raffinose oligosaccharides (raffinose, stachyose, and verbascose). These oligosaccharides are derivatives of sucrose that contain one or more α-D-Galp residues.
Starch is an abundant branched polysaccharide that is the dominant carbohydrate energy storage form in green plants. Starch in grains, tubers, and fruit accounts for most of the calories that humans consume directly as food or indirectly from livestock-fed plants. Starch consists entirely of glucose and exists as amylopectin and amylose. Amylopectin is a branched polymer comprising α-1-4 and 1-4-6-linked D-Glcp, whereas amylose is a linear polymer composed of only α-1-4-linked Glcp. Starch polymers are arranged in insoluble, semicrystalline granules in chloroplasts or specialized plastids (amyloplasts).
Plants also produce fructose-containing polysaccharides termed fructans. The simplest fructan is inulin, a linear glycan of sucrose with 1-2-linked β-D-Fruf. Other inulins contain two fructan chains on a sucrose core molecule. Levan type fructans in grasses contain linear 2-6-β-D-Fruf polymers. Fructans are often utilized by green plants as alternative or additional storage glycans but are stored in the cell's vacuole. It is believed that fructans are also involved in plant protection—in particular, abiotic stresses including drought, salt, or cold stress.
A substantial portion of the carbohydrate formed by photosynthesis is used to produce the polysaccharide-rich walls that surround plant cells. Primary and secondary cell walls are distinguished by their composition, architecture, and functions. A primary wall surrounds growing and dividing plant cells and nongrowing cells in the soft tissues of fruits and leaves. These walls are capable of controlled extension to allow the cell to grow and expand but are sufficiently strong to resist the cell's internal turgor pressure. A much thicker and stronger secondary wall is often formed once a cell has ceased to grow. This secondary wall is deposited between the plasma membrane and the primary wall and is composed of layers that differ in the orientation of their cellulose microfibrils, the type of hemicellulose, the amount of pectin, and the frequent incorporation of the hydrophobic, noncarbohydrate, polyphenolic polymer lignin. For example, the secondary walls of vascular tissues involved in the movement of water and nutrients through the plant are further strengthened by the incorporation of lignin. The ability to form conducting tissues with lignified and rigid secondary walls was an indispensable event in the evolution of vascular land plants, as it facilitated the transport of water and nutrients and allowed extensive upright growth and thus a competitive advantage for the exposure to and capture of sunlight. Secondary cell walls account for most of the carbohydrate in plant biomass, such as straw for animal feed and wood for the production of paper and lumber for construction. Because of its abundance, plant biomass is also considered as a renewable, carbon-neutral feedstock for the production of biofuels, biomaterials, and other value-added commodity chemicals (Chapter 59).
Primary cell walls are composites that resemble fiber-reinforced porous, aqueous gels. The complex structures and functions of these walls result from the assembly and interactions of a limited number of structurally defined polysaccharides and proteoglycans. Nevertheless, wall structure and organization differ between plant species and in different tissue and cell types within a plant. Moreover, during cell division and differentiation, and in response to biotic and abiotic challenges, a cell often responds by the differential synthesis and modification of the noncellulosic components or by the addition of new components.
Primary walls of land plants contain cellulose, hemicellulose, and pectin, in different proportions. They also contain structural proteins/proteoglycans, enzymes, low-molecular-weight phenolics, and minerals. Pectin and hemicellulose are present in approximately equal amounts in the so-called type I primary walls of gymnosperms, dicots, and nongraminaceous monocots, whereas hemicellulose is far more abundant than pectin in the type II walls of the grasses. Recent studies of walls from diverse land plants and cell type–specific characterization of wall glycans indicate that the breadth of wall composition and structural diversity is enormous and thus one should think of walls as a structural continuum rather than specific types.
Cellulose, the most abundant biopolymer in nature, is a linear glycan composed of 1-4-linked β-D-Glcp residues (Figure 24.1A). Several of these chains are hydrogen bonded to one another to form paracrystalline microfibrils. Each microfibril is predicted to contain between 18 and 24 glucan chains. The glucan chain is synthesized by a cellulose synthase complex at the cell's plasma membrane. Three cellulose synthases, encoded by three different genes, are believed to interact to form a trimeric complex, which in turn assembles into a hexameric rosette at the plasma membrane. The catalytic site of each cellulose synthase is located in the cytosol and transfers glucose from UDP-Glcp onto the elongating glucan chain. The mechanisms involved in the formation of a microfibril from individual glucan chains are not well understood, although it may involve an assembly process that is facilitated by specific proteins. The newly formed microfibrils are deposited in the wall of a growing cell with an orientation that is transverse to the axis of elongation. This orientation may be guided in part by protein-mediated interactions between cellulose synthase proteins and cortical microtubules.
Many properties of native cellulose depend on interactions that occur at the surface of the microfibrils. The surface chains are accessible and reactive, whereas the hydroxy groups of the internal chains in the crystal participate in extensive intra- and intermolecular hydrogen bonding. Cellulose is insoluble in water and somewhat resistant to hydrolysis by endo- and exoglucanases because of this highly packed arrangement of the glucan chains.
A number of organisms including fungi and bacteria are specialized in depolymerizing cellulose. This involves several types of enzymes including endoglucanases, cellobiohydrolases, and β-glucosidases. Many of these enzymes have a catalytic domain connected to a cellulose-binding module. This module facilitates binding of the enzyme to the insoluble substrate. Some microorganisms also produce copper-dependent oxidases that render crystalline cellulose more susceptible to hydrolysis. Cellulases and other enzymes involved in cellulose hydrolysis often exist as macromolecular complexes referred to as cellulosomes. Improving the effectiveness of cellulosomes is an area of active research, to increase the conversion of plant biomass to fermentable sugar (Chapter 59).
Hemicelluloses are branched polysaccharides with a backbone composed of 1-4-linked β-D-pyranosyl residues with an equatorial O-4 (Glcp, Manp, and Xylp). Xyloglucan, glucurono/arabinoxylan, and glucomannan (Figure 24.1B–E) are included under this chemical definition of hemicelluloses. Hemicelluloses and cellulose have structural and conformational similarities that allow them to form strong, noncovalent associations with one another in the cell wall, although the biological significance of these interactions is still a subject of debate.
Xyloglucan and cellulose both have a backbone of 1-4-linked β-D-Glcp residues, but unlike cellulose, xyloglucan contains sidechain substituents. The xyloglucan backbone is highly substituted with α-Xylp substituents at O-6 and in some cases with O-acetyl-substituents (Figure 24.1B). Each Xylp residue may itself be extended by the addition of one or more monosaccharides including β-D-Galp, α-L-Fucp, α-L-Araf, α-L-Arap, β-D-Xylp, β-D-GalpA, and O-acetyl substituents. Twenty-three structurally unique side chains have been identified to date, although only a subset of these are synthesized by a single plant species.
Early models of dicot primary walls predicted that xyloglucans acted as tethers between cellulose microfibrils and that controlled enzymatic cleavage or reorganization of xyloglucan by proteins facilitated wall expansion and thus plant cell growth. However, this notion has been challenged by genetic engineering of xyloglucan structures in Arabidopsis plants, which revealed that eliminating xyloglucan entirely from the primary wall has remarkably little effect on overall plant growth and development. In contrast, removing only selected sidechain substituents of xyloglucan is detrimental to plant growth and replacing the sidechains with various glycosyl moieties restores plant growth, independent of the glycosyl moiety added. These results have led to the suggestion that xyloglucan acts as a spacer molecule to keep cellulose microfibrils apart and that pectin has a more important role in controlling wall expansion than previously believed.
Arabinoxylan (Figure 24.1D) is the predominant noncellulosic polysaccharide in the type II walls of the grasses, with only small amounts present in dicot primary walls. Its backbone is composed of 1-4-linked β-D-Xylp residues, many of which are substituted at O-3 with α-L-Araf residues. These Araf residues may be further substituted at O-2 with an α-L-Araf or a β-D-Xylp residue. A small number of the backbone residues are substituted at O-2 with α-D-GlcpA and its 4-O-methylated counterpart (MeGlcpA).
The presence of 1-3, 1-4-linked β-glucans (also referred to as mixed-linkage glucans) (Figure 24.1F) in the walls of grasses was once considered to be a unique feature of these plants. However, structurally related mixed-linkage β-glucans have also been identified in the walls of Selaginella (lycopod) and Equisetum (horsetails), although the evolutionary relationship between these β-glucans is not known. In grasses, mixed linkage glucan is present mainly in young tissues, which has led to the suggestion that it is a carbohydrate storage molecule as also indicated by its metabolism during plant development.
Callose, a polysaccharide composed of 1-3-linked β glucosyl residues, is another β-glucan produced by plants (Figure 24.1G). It is used to form a temporary cell wall at the cell plate during cytokinesis and is involved in regulating the permeability of plasmodesmata. It is often formed in response to abiotic and biotic stresses or damage.
Pectins are structurally complex polysaccharides that contain 1-4-linked α-D-GalpA. Three structurally distinct pectins—homogalacturonan, substituted galacturonan, and rhamnogalacturonan—have been identified in plant cell walls (Figure 24.2). Homogalacturonan, which may account for up to 65% of the pectin in a primary wall, is composed of 1-4-linked α-D-GalpA. The carboxyl group may be methyl-esterified, thus neutralizing the negative charge of this glycosyl moiety, and the uronic acid itself may be acetylated at O-2 or O-3. The extent of methyl-esterification is controlled by pectin methylesterases and pectin methylesterase inhibitors present in the wall. The degree of methylesterification affects the ability of homogalacturonan-containing glycans to form ionic calcium cross-links with themselves and with other pectic polymers. Such interactions alter the mechanical properties of the wall and may influence plant growth and development.
Rhamnogalacturonan-I (RG-I) is a family of polysaccharides with a backbone composed of a repeating disaccharide 4-α-D-GalpA-1-2-α-L-Rhap-1. Many of the GalpAs are acetylated at O-2 and/or O-3. Depending on the plant, between 20% and 80% of the Rhap residues may be substituted at O-4 with linear or branched side chains composed predominantly of Araf and Galp, together with smaller amounts of Fucp and GlcpA (Figure 24.2). Little is known about the functions of these side chains and their contribution to the properties of the primary wall.
Substituted galacturonans have a backbone composed of 1-4-linked α-D-GalpA acid residues that are substituted to varying degrees with mono-, di-, or oligosaccharides. For example, xylogalacturonans contain single β-D-Xylp residues linked to O-3 of some of the backbone residues (Figure 24.2), whereas apiogalacturonans have β-D-apiose (Apif) and apiobiose linked to O-2 of some of the backbone residues. Apiogalacturonans have only been identified in the walls of duckweeds and seagrasses.
The substituted galacturonan referred to as RG-II, which accounts for between 2% and 5% of the primary cell wall, is the most structurally complex polysaccharide yet identified in nature. It is composed of 12 different monosaccharides linked together by up to 21 distinct glycosidic linkages (Figure 24.2). Four structurally different side chains and one or two Araf substituents are attached to the galacturonan backbone. Two structurally conserved disaccharides (side chains C and D) are linked to O-3 of the backbone. The A and B side chains, which contain between 7 and 9 monosaccharides, are linked to O-2 of the backbone. Several of the monosaccharides in RG-II are O-methylated and/or O-acetylated.
Virtually all of the RG-II exists in the primary wall as a dimer cross-linked by a borate ester. The ester is formed between the Apif residue in side chain A of each RG-II monomer (Figure 24.2). The dimer forms rapidly in vitro when the RG-II monomer is reacted with boric acid and a divalent cation. However, the mechanism and site of dimer formation in planta has not been determined. Borate cross-linking of RG-II is likely to have substantial effects on the properties of pectin and the primary wall as RG-II is itself linked to homogalacturonan (Figure 24.2). Indeed, mutations that affect RG-II structure and cross-linking result in plants with abnormal walls and severe growth defects. Swollen primary walls and abnormal growth together with reduced RG-II cross-linking are also a characteristic of boron deficient plants. RG-II was believed to be largely resistant to fragmentation by microbial enzymes. However, recent studies have shown that bacteria present in the human gut produce glycanases capable of hydrolyzing all but one of the glycosidic bonds in RG-II.
Pectin is believed to exist in the cell wall as a macromolecular complex comprised of structural domains—homogalacturonan, rhamnogalacturonan, and substituted galacturonan—that are covalently and noncovalently linked to one another. However, there is only a limited understanding of how these structural domains are organized (Figure 24.2). Molecular modeling of a pectin (∼50 kDa) containing homogalacturonan (degree of polymerization ∼100) and rhamnogalacturonan with arabinogalactan side chains, together with modeling of RG-II conformation have begun to provide insights into the conformations and relative dimensions of each pectin structural domain.
The homogalacturonan region has a persistence length of approximately 20 GalAp residues, which is likely to be sufficient to stabilize junction zones formed with Ca++. In vitro studies suggest that the maximum stability of such junction zones is obtained with oligomers containing approximately 15 nonesterified GalpA residues. Thus, controlling the distribution of methyl-ester groups along the homogalacturonan backbone provides a mechanism to regulate the physical properties of pectin, including its ability to form gels. For a gel to form and not to be brittle, other features including sequences that interrupt interchain associations in the pectin macromolecule may be important. For example, the structural diversity and the conformational flexibility of the oligosaccharide side chains of the rhamnogalacturonan domain will limit or prevent interchain pairing. The presence of 1-2-linked Rhap residues does not introduce kinks into the backbone geometry of rhamnogalacturonan and thereby limit interchain associations. Rather, it is the side chains linked to these residues that are responsible for preventing or limiting interchain associations.
The conformations of the four side chains attached to the homogalacturonan backbone may lead RG-II to adopt a “disk-like” shape. Well-defined tertiary structures are predicted for the RG-II monomer and the dimer. In the dimer, borate-ester cross-linking and Ca++ interchain pairing further stabilizes the two disks. The apparent resistance of RG-II to wall-modifying enzymes together with the formation of a cation-stabilized RG-II dimer likely results in a structure that resists temporal changes in the plant. In contrast, homogalacturonan is continually modified by the action of wall enzymes and its contribution to wall architecture is therefore time dependent.
Increased knowledge of the physical properties of primary wall polysaccharides and proteoglycans is required to understand how modulating the amounts and structural features of a few common polysaccharides and glycan domains lead to primary walls with diverse properties and functionalities. Further research is also needed to determine if wall structure and function results from the noncovalent interactions of polysaccharides and proteoglycans or from the formation of glycan-containing architectural units with specific structural and functional roles. The latter scenario is analogous to the organization of proteoglycans and O-linked mucins in the extracellular matrix of animal cells (Chapters 10, 16, and 17).
The secondary walls of woody tissue and grasses are composed predominantly of cellulose, hemicellulose, and the polyphenol lignin. The inclusion of lignin results in a hydrophobic composite that is a major contributor to the structural characteristics of secondary walls.
Heteroxylans are the major hemicellulosic polysaccharide present in the secondary (lignified) cell walls of seed-producing plants. These heteroxylans are classified according to the type and abundance of the substituents on the 1-4-linked β-D-Xylp residues of the polysaccharide backbone. Glucuronoxylans, which are major components in the secondary walls of woody and herbaceous eudicots, have an α-D-GlcpA or MeGlcpA substituent at O-2 (Figure 24.1C). Gymnosperm secondary walls contain arabinoglucuronoxylans (AGXs), which in addition to MeGlcpA substituents, have Araf residues attached to O-3 of some of the backbone residues. The glucuronoarabinoxylans in the secondary walls of grasses typically contain fewer Araf residues than their primary wall counterpart (Figure 24.1D). Ferulic or coumaric acids are often esterified to the Araf residues of xylan in grass primary and secondary cells walls. There is some evidence that the lignin polymer is covalently linked to the secondary wall hemicelluloses.
Eudicot and gymnosperm secondary wall xylans have a well-defined glycosyl sequence 1-4-β-D-Xylp-1-3-α-L-Rhap-1-2-α-D-GalpA-1-4-D-Xylp at their reducing end (Figure 24.1C). This sequence is required for normal xylan synthesis during secondary cell-wall formation and may have a role in regulating the polymers chain length. This sequence is present at the reducing end of heteroxylans of all monocots except the grasses.
Genes that encode polysaccharide biosynthetic enzymes, including many of those required for xyloglucan, glucuronoxylan, arabinoxylan, and cellulose synthesis and some of those required for pectin synthesis, have been identified. This information, together with improved methods to demonstrate enzymatic activity of recombinant plant GTs in vitro and wall structural analyses of the corresponding plant mutants is providing a framework for an increased understanding of how plant cell wall polysaccharides are synthesized. Most pectins and hemicellulose are synthesized in the Golgi apparatus and then secreted into the apoplast via vesicles. This is in contrast to cellulose, which is synthesized at the plasma membrane where the glucan chains are extruded into the apoplast to form cellulose microfibrils. Lignin is polymerized in the apoplast by nonenzymatic radical reactions. Despite advances in identifying and understanding the GTs involved in polysaccharide synthesis, we still do not know how many of the wall polymers are synthesized by Golgi-localized multienzyme complexes or if they are assembled by GTs localized in different regions of the Golgi apparatus. We also do not understand how the newly synthesized polymers are assembled into a functional cell wall in the apoplast.
Plants produce glycoproteins and proteoglycans that contain oligo- or polysaccharides that are linked to hydroxyproline (Hyp) and serine (Ser). The protein component of these is present in relatively low abundance in the wall. Hyp is formed posttranslationally by endoplasmic reticulum (ER)-localized prolyl hydroxylases and is O-glycosylated in the ER and in the Golgi apparatus. The degree and type of Hyp glycosylation is determined to a large extent by the protein's primary sequence and the arrangement of Hyp residues. Hyp glycosylation is initiated by the addition of an Araf or a Galp residue. Contiguous Hyp residues are arabinosylated, whereas clustered but noncontiguous Hyp residues are galactosylated. Ser residues and occasionally threonine residues may also be O-glycosylated in these proteins.
Three classes of structurally distinct plant proteoglycans containing glycosylated Hyp and Ser—the extensins, proline/hydroxyproline-rich proteoglycans, and arabinogalactan proteins—have been identified. Extensins are hydroxyproline-rich proteoglycans with Ser(Hyp)4 repeat sequences and contain between 50% and 60% (w/w) glycan. Most of the carbohydrate exists as oligosaccharides containing one to four Araf residues linked to Hyp together with a small number of single Galp residues α-linked to Ser. The genetic modification of extensin glycosylation levels resulted in the blockage of polarized cell growth in root hairs, underpinning the importance of extensin glycosylation in plant growth and development. The proline/hydroxyproline-rich proteoglycans, which contain from 3% to 70% (w/w) carbohydrate, are distinguished from the extensins by amino acid sequence. Both of these families of hydroxyproline-rich glycoproteins (HRGPs) likely have a structural role in the cell wall. The expression of genes involved in their synthesis is developmentally regulated and is often induced by wounding and fungal attack of plant tissues. Various plant glycopeptide signaling molecules including clavata contain arabinosylated hydroxyproline and have numerous roles in plant growth and development.
Arabinogalactan proteins (AGPs) have a glycan content of up to 90% (w/w). Chains of between 30 and 150 monosaccharides are linked to the protein by Galp-O-Ser and Galp-O-Hyp linkages. These chains have a 1-3-linked β-Gal backbone that is extensively substituted at O-6 with side chains of 1-6-linked β-Galp. These side chains are terminated with Araf, GlcAp, and Fucp residues. Some AGPs may contain homogalacturonan, RG-I, and xylan covalently linked to the arabinogalactan (Figure 24.3), thereby forming a protein–hemicellulose–pectin complex referred to as APAP1. The location of this complex in the plant and its biological function remains to be determined.
Several AGPs are secreted into the cell wall, whereas others are linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Plant GPI anchors contain a phosphoceramide core. The glycan portion of the GPI anchor of pear cell AGP has the sequence α-D-Manp1-2α-D-Manp-1-6-α-D-Manp-1-4-GlcpN-inositol. At least 50% of the Manp attached to the GlcpN (see Chapter 12) is itself substituted at O-4 with a β-Galp, a feature that may be unique to plants. Many functions have been proposed for the AGPs including their participation in signaling, development, cell expansion, cell proliferation, and somatic embryogenesis.
Many of the proteins that have passed through the plant secretory system contain N-linked oligomannose, complex, hybrid, or paucimannose-type glycans (Figure 24.4). The initial stages of the synthesis of these N-glycans, including the transfer of the oligosaccharide precursor from its dolichol derivative and the control of protein folding in the ER, are comparable in plants and animals (Chapter 9). However, two modifications of N-glycans during passage through the Golgi are unique to plants.
Oligomannose-type N-glycans are often trimmed in the cis-Golgi and then modified in the medial-Golgi by N-GlcNAc transferase I (GnT-I) catalyzed addition of GlcNAc to the distal Man of the core. In reactions that are typical to plants, a β-Xylp is often added to O-2 of the core Manp. In the trans-Golgi, α-Fucf may be added to O-3 of the GlcpNAc residue that is itself linked to asparagine (Figure 24.5). The XylT and FucT that catalyze these reactions act independently of one another but do require at least one terminal GlcpNAc residue for activity. The FucT is related to the Lewis FucT family, whereas the XylT is unrelated to other known GTs.
Processing of N-glycans in the plant secretory system. Only those events that are unique to plants are shown in detail.
The xylosylated and fucosylated N-glycans are often trimmed by α-mannosidase II. A second GlcNAc may then be added by GnT-II. Some plant N-glycans do not undergo further mannose trimming and proceed through the Golgi as hybrid-type N-glycans. Complex and hybrid-type N-glycans may be further modified by the addition of Galp and Fucp in the trans-Golgi. Plant glycoproteins are either secreted from the cell or transported to the vacuoles. Many of the glycoproteins present in the vacuoles contain paucimannose type glycans, suggesting that they are trimmed by vacuolar glycosidases (Figure 24.5).
The presence of sialic acid in the N-glycans of plant glycoproteins was claimed but likely represented environmental contamination. Plants do have genes that encode proteins containing sequences similar to sialyltransferase motifs, but their functions have not been established.
Plant-specific modifications of N-glycans result in glycoproteins that are often highly immunogenic and cause allergic responses in humans. The demonstration that complex N-glycans are not essential for plant growth initiated studies to engineer plant N-glycosylation pathways to produce glycoproteins that do not activate the mammalian immune system. Plants lacking the GTs that add Xylp and Fucp to N-linked glycans produce glycoproteins lacking immunogenic glyco-epitopes. Other glycosylation pathways involved in the addition of sialic acid and Galp must be introduced to fully “humanize” the glycoproteins if plants are to be used to produce recombinant therapeutic glycoproteins.
Only a few glycans of green algae in the Chlorophyta and Streptophyta clades have been studied in detail. However, understanding their diverse polysaccharide structures could have important implications for the evolution of the more complex structures present in land plants.
The Viridiplantae are believed to have diverged into the Chlorophyta and Streptophyta, between 800 and 1200 million years ago. The Chlorophyta, which include diverse marine, freshwater, and terrestrial green algae, often have cell walls that are quite distinct from the walls of the Streptophytes. For example, the extracellular matrix of the chlorophyte Chlamydomonas reinhardtii is a crystalline lattice formed from HRGPs, whereas this matrix is rich in Kdo and Dha in the prasinophytes.
The Streptophyta comprise land plants and the charophyte algae including the late diverging Zygnematophyceae, which are currently believed to include the closest living relatives of land plants. Indeed, the cell wall of Penium margaritaceum, a unicellular Zygnematophyte, contains cellulose, pectic, and hemicellulose-like glycans.
With the emergence of whole-genome sequencing and extensive transcriptomic analyses, detailed phylogenetic trees of glycan synthesis-related GTs are being established including GTs from green algae and land plants. Such data are required to develop hypotheses predicting when and how different structural forms of diverse cell wall polysaccharides evolved across the Chlorophyta and Streptophyta. Detailed structural and architectural analyses of algal and land plant cell wall glycans and the substrate and acceptor specificities of the corresponding GTs will be required to resolve many of the evolutionary transitions within the Viridiplantae.
Numerous specialized algal polysaccharides are used by humans. For example, several of the polysaccharides produced by red and brown algae are used in the food industry as gelling agents, stabilizers, thickeners, and emulsifiers. They are also used in paints, in cosmetics, in paper, and as reagents for scientific research. These polysaccharides include agarose (agar) and carrageenan, which are sulfated galactans obtained from red seaweeds. These polysaccharides are composed of the repeating disaccharide 3-β-D-Galp-1-4-3,6-anhydro-α-L-Galp-1 unit. Some of the d-Galp and l-Galp units are O-methylated. Pyruvate and sulfate groups may also be present in small quantities. Alginate, a linear polysaccharide composed of 1-4-linked β-D-ManpA and its C-5 epimer 1-4-linked α-L-GulpA, produced by various species of brown seaweed is another example of a commercially important polysaccharide. These monosaccharides are typically arranged in blocks of either ManpA or GulpA separated by regions comprised of 4-ManpA-1-4-GulpA-1 sequences. Brown seaweeds produce polysaccharides that have potential in the treatment of diseases. Laminaran is a linear storage polysaccharide composed of 1-3- and 1-6-linked β-D-Glcp residues. There are reports that laminaran has antiapoptotic and antitumor activities. Fucoidans are a group of sulfated polysaccharides isolated from several brown algae that have been reported to have anticoagulant, antitumor, antithrombosis, antiinflammatory, and antiviral properties. Fucoidans have a backbone of 1-3-linked α-Fucp that is substituted at O-2 with fucose and at O-4 with sulfate or fucose. Other fucoidans have backbones of alternating 1-3- and 1-4-linked α-Fucp residues.
Glycoglycerol lipids are the most abundant glycolipids in plants. Mono- and digalactosyldiacylglycerol have been identified in all plants, whereas tri- and tetragalactosyldiacylglycerol have a more restricted taxonomic distribution (Figure 24.6). The synthesis of these galactolipids is initiated by the formation of diacylglycerol in the ER membrane and the chloroplast membrane. Galactolipids formed in the ER membrane contain predominantly C16 fatty acids at the sn-2 position and C18 fatty acids at the sn-3 position. The chloroplast pathway produces C18 fatty acids at both positions. Each of these fatty acids is then desaturated to 16:3 or 18:3 acyl groups. Monogalactosyldiacylglycerol (MGDG) is synthesized by the transfer of Gal from UDP-Galp to diacylglycerol by an MGDG synthase. Digalactosyldiacylglycerol (DGDG) is formed from MGDG by the transfer of Galp from UDP-Galp by a DGDG synthase. These reactions occur primarily in the outer chloroplast membrane. The products are then transported to the inner membrane and the thylakoid membranes of the chloroplast. The presence and abundance of MGDG in the chloroplast thylakoid membrane are important for normal photosynthesis to occur. Sulfoquinovosyldiacylglycerol, which is formed from diacylglycerol, is also abundant in the thylakoid membrane and may also have a role in photosynthesis.
The most abundant plant galactolipids.
Small amounts of MGDG and DGDG are present in the cytosolic leaflet of the plasma membrane, although the mechanism of galactolipid exchange among the membranes is not understood. The outer leaflet of the plasma membrane is instead dominated by glycosylinositolphosphorylceramides (GIPCs), lipids that are absent in animals. GIPCs were first described in the 1950s, but have remained poorly characterized partly because of their insolubility using standard membrane extraction protocols. GIPCs are estimated to comprise up to 40% of the plasma membrane, with more than 200 species described. Ceramide is synthesized in the ER and is then transported to the Golgi where an inositol phosphate and several glycosyl residues are added. The first sugar added in flowering plants is GlcpA, but after that the identity of the sugar, the number of sugars, and their degree of branching depend on tissue and species. GIPCs containing from two (Series A) to seven (Series F) sugars, including Manp, Glcp, GlcpN, GlcpNAc, Galp, and Araf, have been consistently identified, although there have been reports of GIPCs containing up to 20 sugars. Further improvements to the isolation and characterization methods are required so that the full array of glycan structures can be described. No gangliosides have been identified in plants. Reports that Kdop-containing lipids with homology to bacterial lipid A are present in plant organelle membranes remain to be confirmed.
GIPC glycosylation mutants can be lethal or have severe developmental phenotypes. Altering the glycan structure can induce severe constitutive defense responses, alter plant–microbe interactions (both pathogens and beneficial microbes), and reduce the cellulose content of the cell wall. Only a limited number of GIPC GTs have yet been described, and much work is required to fully describe their function.
Plants produce numerous phenolics, terpenes, steroids, and alkaloids that are collectively referred to as secondary metabolites. Many of these compounds are O-glycosylated or contain sugars linked via N, S, or C atoms. Glycosylated secondary metabolites often have important roles in a plant's response to biotic and abiotic challenges and may also have value as pharmaceuticals.
In general, the addition of a single sugar or an oligosaccharide may increase water solubility, enhance chemical stability, or alter both chemical and biological activity. For example, the activity of several plant hormones may be regulated by converting them to their glucose esters or their glucosides. Digoxin and oleandrin are potent cardiac glycosides isolated from foxglove and oleander, respectively. Myrosinase-catalyzed cleavage of S-linked Glcp from glucosinolates leads to the formation of pungent mustard oils when mustard and horseradish are damaged. The steviol glycosides, which are far sweeter than sucrose, are used as natural sugar substitutes. The bitter taste of citrus fruits is due to naringin, a glycosylated flavonoid.
The authors appreciate helpful comments and suggestions from Todd Lowary, Katharina Paschinger, and Iain B.H. Wilson and thank Bernard Henrissat for help with the CAZy numbers in Table 24.1.
The content of this book is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view the terms and conditions of this license, visit https://creativecommons.org/licenses/by-nc-nd/4.0/
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