Plant Cell Wall – Structure & Biosynthesis

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Plant Cell Wall – Structure & Biosynthesis

in our environment we are surrounded by plants our most abundant renewable resource my name is Deborah moanin and in this presentation Melanie at mojo and I from the complex carbohydrate Research Center at the University of Georgia will summarize our current understanding of plant cell wall structure and synthesis each year approximately 100 billion tons of carbon dioxide are fixed into biomass with roughly 1/3 of that produced by marine plants and microorganisms and 2/3 from land plants in photosynthesis plants use carbon dioxide and water taken up from the environment and energy from the Sun to produce carbohydrates the bulk of that fixed carbon ends up in the plant cell walls plant cell walls are the carbohydrate rich extracellular matrix that surrounds all plant cells plant cell walls and plant cells vary in shape and structure depending upon the cell type as can be seen in these micro graphs note the difference in shape and surface structure of the epidermis of a rabid abscess sepals the leaf like structure that supports petals on the upper left versus the epidermis of petals on the upper right furthermore note the intricate branch structure on the bottom-left of single cell trichomes versus the elongate and pitted structure of pollen cells on the bottom right finally note the round structure of suspension cultured cells grown in liquid culture on the bottom middle versus the rigid and thicker wall structure of the large xylem cells which make up water transporting vascular cells and cells in wood plant cell walls have diverse and critical functions in the plant including providing structure to the plant and plant cells being involved in plant growth giving the plant flexibility as you can see when plants sway in the wind they provide hydration as you may see when a seed germinates they are a reservoir of defense and signaling molecules and they provide cell adhesion between a Jason sells and are involved in development if you look at the picture what you will see is the wild-type rep adopts us plant on the Left compared to a mutant plant that is mutant in one of the plant cell wall biosynthetic enzymes clearly showing that a knockdown and expression of a wall bio synthetic enzyme can lead to dwarfism plant cell walls also have many uses for humans and animals they provide clothing wood and lumber products they’re used for bio materials including nano composites nano cellulose fibers biofuels and chemicals they’re also used for animal and human food and fiber and as gelling and stabilizing agents in the food industry and finally cell walls have uses as nutraceuticals and pharmaceuticals so what are plant cell walls plant cell walls are extracellular matrices that are comprised of 80 to 90 percent carbohydrate and approximately 10% protein some cell walls also contain lignin there are generally two types of plant cell walls the primary wall versus the secondary wall in the middle picture here you can see an example of a secondary wall this is a cross-section of wood the wall is thicker if you look on the right however you see the primary wall the primary wall is the first wall laid down around all dividing and growing cells after that wall is produced some cells go on to produce a secondary wall and that primary wall is pushed outward on the right hand side you see seedlings underneath callus culture and then finally suspension cultured cells all of these cells consists largely of cells made up a primary wall there are two types of primary wall dicots and none grass monocots have a so-called type 1 wall as a rabid abscess on the bottom left the grasses have a so-called type 2 wall now finally if you look at the bottom picture you’ll see a cross-section of four contiguous cells you have the plasma membrane and outside the plasma membrane is the primary wall and if you look at this primary wall already at this level of viewing you can see that it is fibrous the wall that surrounds plant cells is cell type developmental state and to some extent species specific so let’s review primary walls are the first wall laid down they surround meristematic and growing cells they’re prevalent in the succulent parts of the plant they’re found at the junction between cells and they form the outer layer even of cells that have secondary walls they’re composed of roughly 90% carbohydrate and 10% protein

and there are two types type one and type two primary walls secondary walls surround cells that differentiate to form specialized functions like wood cells xylem cells and fiber cells they have a different polysaccharide composition than primary walls and they are often lignified plant cell walls consist of interacting polymers if we look at an electron micrograph of a type one wall on left versus a type 2 wall middle top we see that both type of walls contain long fibers that interact and there are also some globular structures now over the last 40 or 50 years carbohydrate chemists have defined many of the structures in the cell wall and we’re beginning to understand wall architecture but we have a lot to learn most models of the plant cell wall will depict three types of polysaccharide matrices the fossil osore cellulose fibers in green Hemi cellulose which is a family of polysaccharides here shown simply in red which in part hydrogen bond to cellulose and then moved into the matrix and the other matrix polysaccharide besides how my cellulose pectin which here are shown as simple ribbons but which we’ll see shortly are much more complex all three types of polysaccharides are present in both type 1 and type 2 primary walls and in secondary walls however their relative amounts differ in the different types of walls type one primary walls are abundant in pectin with 30 to 35 percent pectin and contain less but substantial amounts of the other polysaccharides with 20 to 30 percent cellulose and 25 to 30 Hemi cellulose however the type 2 primary walls of grasses contain much less pectin ranging from two to ten percent depending on the cell type and therefore have higher amounts of cellulose and hemicellulose secondary walls also have less pectin and increased amounts of cellulose and hemicellulose and are often lignified multiple models for the plant cell wall had been proposed over the past 40 years or more however it is important to note that all current models are hypothetical in fact interestingly in their recent book entitled plant cell walls the authors who combined have more than 150 years of research experience in walls decided not to depict an integrated model of the wall but rather depicted characteristics of the different types of matrices in this talk we will mostly use the same strategy however to afford some conceptual framework for you we will first show a very schematic overview of the wall and then provide to individual representations of type one and type two primary walls the generally accepted model of the plant cell wall is based on a cellulose matrix polysaccharide network here you see a depiction of cell one its plasma membrane beneath that its primary wall then the middle lamella and then the primary wallet cell 2 and finally the plasma membrane of cell 2 in the cell wall there are cellulose fibers and interspersed between those fibers not pictorially shown here but demonstrated in words are the matrix polysaccharides Hamas Aiello’s and pectin one point to make is that cell walls consists of a cellulose matrix polysaccharide network that resists tension this aspect of the wall is often attributed to the cellulose hemicellulose network but walls also resist compression and searing forces and that aspect of the wall is often attributed to the pectin Network finally that junction between the cells the middle lamella is actually the very first part of the wall laid down when cells divide by cytokinesis and this pectin rich most plant cell wall models represent the three types of polysaccharide cellulose hemicellulose and pectin but do not represent the protein even though wall protein makes up about 10% of the wall this is the only set of slides in this talk where I’ll show a bit more detail in these cell wall models what I want to point out and make sure you remember is that all wall models at this point are hypothetical what we have here is a type one primary wall on the left and a type two primary wall on the right both have cellulose fibers both have hemi cellulose is partly hydrogen bonded to the cellulose but we see now in more detail and illustration of the pectins if we look on the left you’ll see the pectins in yellow have linear portions and branched portions and if we look on the right you can see this ionic interactions between some of these regions but what about the cell wall proteins cell wall proteins constitute five to ten percent of cell wall mass most cell wall proteins are basic

proteins and that makes sense because the wall contains a lot of negatively charged pectin and some other polymers most cell wall proteins are post translationally modified by one or more of the following hydroxylation of prolene this is often common in wall structural proteins not only in plants but in animal systems and glycosylation ol glycosylation and glycosyl phosphatidyl inositol anchors are so called GPI anchors there are many different types of cell wall proteins for example in a rabid abscess there are 166 wall hydroxyproline rich glycoproteins these include 85 so called a rabinal Galacta proteins which are highly glycosylated and we will come back and look in detail at some so-called a GPS later there are also 59 so-called extensions which are cell wall hydroxyproline which proteins that are moderately glycosylated and finally there are eight prolene rich glycoproteins which are more lightly glycosylated it has been difficult to ascribe specific function to the group of a GPS a Rabbinical actin since they may be soluble gpi-anchored and or located throughout the wall with heterogeneity in protein core structure like oscillation and cell type expression however different a GPS have been shown to be essential for diverse aspects of plant growth and development so we know they play important and necessary rules extensions another type of hydroxyproline rich wall protein are believed to have mechanical functions in the wall and at least for extension three it has been proposed the function as a self-assembling anthe fill important for wall assembly although biological confirmation of this function has been difficult to achieve new results show that proteoglycans that connect the proteome of the wall to the polysaccharides in the wall exist in plant cells however how prevalent in diverse these new type of proteoglycans are is just now beginning to be investigated the only structurally defined proteoglycan identified to date is a pop one which stands for arabinose island pectin a rabinal actin protein one what is new about a pop one is that you have an Arab on ogle actin protein here shown by the AG P the protein part and attached to that the Arab oh no galactic lichen domain that had been known for some time what was recently shown is that attached to that a rabinal act and domain is a hemi cellulose domain silent one and then on a different region a pectin domain are g1 h gr g1 and then finally attached to that pectin domain is another Hema silos domain xylan to later in the talk we will come back and look at the fine structure of this proteoglycan the existence of a proteoglycan such as a pop one in plant cell walls opens up many questions about wall structure and synthesis for example how ubiquitous is a pop one do other proteoglycans exist in plant walls is a pop one synthesized in the cell or in the cell wall indeed the discovery of a pop one demonstrates that our understanding of wall structure is incomplete in the models shown here we show that a pop one may function as a linchpin in the wall connecting two previously viewed separate types of wall polysaccharides we will now go deeper into wall structure and synthesis as we move from polymer to polymer we will begin by considering a relevant wall structural model then study representative structures and end with an overview of our understanding of the biosynthetic process for each polymer before we begin let’s first look at the big picture where are the wall polymers made and what does that process involve here we see an overview model of plant wall synthesis jeans and cold proteins that exist either in the cytosol to synthesize the wall precursors nucleotide sugars those proteins may exist in the Golgi to synthesize the Hemi cellulose as’ and pectins this would include glycosyltransferase –is that synthesize the polysaccharide backbone and branches and methyl acetyl or other transferases that modified the backbone and finally the proteins may exist as complexes in the plasma membrane to make up the cellulose synthase complex at synthesize cellulose the Hemi cellulose is in pectins are moved in vesicles to the wall fusing at the plasma membrane and in a way we still don’t understand the whole assortment of polymers interact together to make wall final architecture what I just represented was synthesis of the

primary wall if we look at the middle panel for those type of cells that have secondary wall particularly wood forming cells that middle lamella and primary wall are pushed out and secondary walls are produced in wood in fact there are three layers known as the s 1 s 2 and s 3 and if we look at the bottom of this figure one difference during the synthesis of many types of secondary walls that is in addition to the polysaccharides and proteins there is a polymeric structure known as lignin made that originates as monolid Knowles made we believe in the cytosol that then moved through the plasma membrane to the wall we will first discuss briefly the poly phenolic polymer present in many types of secondary walls and then move on to the carbohydrates in the wall lignin is an aromatic heterodimer and secondary walls of fibers and water conducting cells it is made by free radical coupling of para hydroxy phenol y asil and syringe l subunits lignin is the second most abundant organic polymer on earth second only to cellulose the water transport tissues in plants contain fibers and xylem cells that are lignified here we see cross-sections of upper and lower stems of medicago lignin is visualized in the top by autofluorescence from the aromatic groups and in the middle by staining with floral gluten all and more lay staining and what you can see is that as the tissues age and you move from younger v internode to older stem sections you get more and more lignin to support those transport tissues lignin is made from three types of mono lignin subunits parakou Merle alcohol coniferous alcohol and sin Appel alcohol which make h g + s lignin the mono lignans are made in the cell transported in a still uncertain mechanism across the plasma membrane and then memorized in the wall by oxidative polymerization since lignin is produced by combinatorial free radical coupling the resulting polymer in the wall contains a series of ether and other linkages in no specific pattern h + g lignin are deposited early during lignin of the middle lamella and cell junctions while g lignin and s lignin are present in xylem vessels and fibers s lignin is present largely in fibers it is believed that plants evolved the ability to synthesize lignin at the point in time when they arose from an aqueous environment and colonized terrestrial environments the enzymes in ways involved in the synthesis of the monolid nulls which are the precursors for lignin are known in great detail and we now have the ability to modify the expression of these enzymes to modify lignin in plants this increases our ability to access the polysaccharides in the wall and is being actively studied in regards to biomass utilization for biofuel and biomaterials we will now summarize our knowledge of cell wall polysaccharides structure and synthesis to do this we will use the same monosaccharide nomenclature as used in the mammalian and animal glycobiology community which originated from the Consortium for functional glycomics just as an example if we look at the illustrations on the top left each shape and color depicts a specific type of monosaccharide a blue circle is glucose a yellow circle is galactose if we look to the right a star is silos the use of these symbols and colors across different types of fields allows facile communication of polysaccharide structure as mentioned previously the precursors for cell wall polysaccharide synthesis include at least 16 different nucleotide sugars that are synthesized largely from photosynthate derived UDP glucose or gdp monos via the nucleotide sugar into conversion pathway for example UDP glucose which is shown at the middle top in this figure is converted to UDP glucan ik acid also called UDP Kluge via oxidation of a hydroxyl to the carboxylic acid furthermore UDP xylose is produced from UDP Klug a via decarboxylation the inter conversion pathway includes epimer ization decarboxylation dehydration oxidation reduction and isomerization reactions some sugars are also produced by phosphorylation and eurid elation or guan elation of monosaccharides recycled

from the wall via the salvage pathway these sugars are shown in the gray ovals in the figure the nucleotide sugar biosynthetic reactions occur largely in the cytosol although several reactions also occur in the Golgi we will begin our overview of cell wall polysaccharides structure and synthesis with cellulose before we look at the detailed structure of cellulose let us consider it within the context of the plant cell wall although each individual cellulose molecule is a simple linear homo polymer of glucose the individual chains interact to form microfibrils and that is the form of cellulose represented in this model this simplified model depicts two adjacent cells with the plasma membrane of cell one at the top followed by the cell wall then the middle lamella connecting the two primary walls then the primary wall of cell 2 and finally the plasma membrane of cell 2 at the bottom the cellulose microfibrils in the primary wall interact via hydrogen bonding with unbranched surfaces of hemi cellulose to yield the so called cellulose hemicellulose network which is shown in this slide this cellulose hemicellulose network is believed to contribute significantly to cell wall mechanical strength so what is cellulose cellulose is the most abundant organic polymer on earth it makes up about 20 to 30 percent of higher plant primary walls and up to 50 percent of secondary walls cellulose is a linear polymer of glucose connected via beta one four linkages these beta animerica lenka jiz result in each glucose residue being flipped 180 degrees yielding a flat linear chain adjacent cellulose chains interact via hydrogen bonding hydrophobic interactions between the flat surfaces of the paranal sugar rings and van der Waals interactions these interactions result in crystalline cellulose microfibrils or fibers the length of the microfibrils vary the degree of polymerization or DP in primary walls has been shown to fall into two classes shorter lengths of 50 to 500 DP and longer lengths of 2,500 to 4,000 the degree of polymerisation of secondary wall cellulose is larger ranging from 10,000 to 15,000 sugar units current data indicate that plant cellulose microfibrils consists of 24 to 36 gluten chains with a diameter of 2 to 4 nanometers and being several microns long the microfibrils may spontaneously associate into macro fibrils or bundles as are commonly found in plants secondary walls cellulose as synthesized in the plant exists as glucan chains arranged parallel to each other this contrasts to industrial process cellulose in which the glucan chains may be arranged anti parallel to each other cellulose is synthesized by a family of proteins known as cellulose synthesis abbreviated si si si si genes exist in multi gene families and plants for example rabbits OPS’s has tens si genes sesay proteins appear to function in protein complexes known as rosettes that are present at the plasma membrane the rosettes contain at least three different Sesay proteins to have functional complexes as will describe later in more detail research has shown that some Sesay proteins function predominantly to synthesize primary wall cellulose while others as a family members function in secondary wall synthesis for example a rabid abscess essays 4 7 & 8 catalyze secondary cell wall cellulose synthesis this slide shows a freeze fracture replica of the plasma membrane of tricky Ariela meant cells from zinnia elegans that are synthesizing secondary cell wall cellulose the three circled structures visible on the cytosolic face of the cytosolic leaflet of the plasma membrane show Sesay containing rosette structures in the plasma membrane each rosette appears to contain six subunits the rosette structures have been shown to move in linear tracks in the plasma membrane that align with cortical microtubules in the cytosol Sesay proteins contain multiple transmembrane domains and cytosolic loop regions involved in catalysis and protein protein interactions as shown in panel B the transmembrane domains form a channel in the plasma membrane through which the growing glucan chains synthesized on the cytosolic side of the plasma membrane are extruded into the wall current models shown in panel C

depict each subunit of the rosette as containing six Sesay proteins if each Sesay protein is catalytically active this would mean that each rosette with simultaneously synthesize 36 glucan chains constituting a microfibril as illustrated in panel d4 primary wall and panel e4 secondary wall available data also indicate that each subunit of the rosette contains sesay proteins encoded by 3 differents SI genes for example a secondary wall rosette would contain sesay proteins 4 7 & 8 we still have much to learn about the mechanism of cellulose synthesis and the exact role of the rosettes in this process the catalytically active bacterial cellulose synthase bcs ABC SB complex roto Bacteroides has recently been crystallized as in plants the catalytic subunit of the bacterial cellulose synthase complex BC sa is a KZ glycosyltransferase family 2 enzyme and the catalytic cytosolic regions of these two enzymes share over 26 percent similarity thus the structure of the bacterial enzyme is likely informative for the plant enzyme the crystal structure of the bacterial cellulose synthase provides a possible answer to the long-standing question of whether cellulose synthesis occurs via a single catalytic site or two that is is the nascent glucan chain extended by one or two lucasz molecules at a time this question arises since each glucose residue in the cellulose chain is flipped 180 degrees relative to the next residue in the chain thus one long-standing proposal has been that there must be two catalytic sites that function iteratively during cellulose synthesis however the crystal structure of the bacterial enzyme supports a single site synthesis mechanism in which synthesis and translocation occur in one site with translocation changing 180 degrees with each addition of glucose we still have much to learn about the complete mechanism for cellulose synthesis in plants it is hypothesized that the Sesay complexes are at least partially assembled in the Golgi and that these complexes are transported to the plasma membrane in Golgi derived vesicles known either as small sesay compartments Smacks or microtubule or associated cellulose synthase compartments masks in addition numerous other proteins have been identified that are believed to be associated with cellulose synthesis these include Corrigan Susie Cobra Cobra like and a series of cytoskeleton related proteins finally as noted earlier in this talk the matrix polysaccharides are known to associate with cellulose particularly hemi cellulose and we don’t know the mechanism of this association an elegant example of the association of cellulose with other wall components is shown by this electron micrograph of untreated cellulose from a corn husk on the left here we see in the natural state that cellulose is associated with waxes lignin pectin and hemi cellulose contrast that view with the partially purified cellulose fibers from the same tissue shown on the right we now discuss the structure and synthesis of the family of matrix polysaccharides known as hemi cellulose the term Hemi cellulose means half cellulose indeed all the Hemi cellulose is with the exception of the mixed linkage glucans have a backbone of beta 1-4 linked glycosyl residues that are flipped 180 degrees to each other yielding regions of patina linear ribbon like surfaces however unlike cellulose the hemi cellulose backbones are branched except for mixed linkage glucans in which the backbone is kinked this branching and kinking prevents the formation of fibers from the hemi cellulose –is furthermore the backbones of the hemi cellulose is contained glucose silos or mannose residues rather than only glucose residues as in cellulose we will now begin our discussion of how my cellulose is by looking in detail at the structure of the hemi cellulose known as silo glucan silo glucan is the major hemi cellulose in dicot and non gram anisha’s monocot primary walls making up about 25 percent of type one primary walls it is also a minor component of type two primary walls as the name would imply xylem glucan has a backbone of beta 1-4 linked glucose residues some of those glucoses are substituted at the sixth position with alpha silos some of the

silos residues are substituted with beta galactose and the beta galactosidase there seems to be a repetitive structure to Xylo glucan in which for every four glucoses within the backbone three of those are frequently silo sedated and furthermore of those three selasa dated side branches two are typically galactose elated and finally one may have a few coats on it a code has been developed as shown in the middle of this slide where a branch for example that has a glucose and xylose on it is called X a free glucose is termed G a branch that has glucose in the backbone xylose and galactose is termed L and a branch that terminates with fucose is termed F there are additional complicated structures for the side branches with additional codes as shown on the left bottom of this figure as indicated earlier cellulose is known to hydrogen bond to Hemi cellulose suggesting that Hemi cellulose must have structural regions or confirmations in which the backbone is available for binding to cellulose molecular modeling of a 17 sugar residue type one primary wall silo glucan structure indeed predicts a so called cellulose like flat glucan backbone conformation adoption of Xylo glucan into this flat glucan backbone conformation would allow hydrogen bonding of silo glucan to cellulose it is important to realize that plants have evolved over hundreds of millions of years to withstand diverse biotic and abiotic stresses by modifying their walls this is believed to be one if not the major driver of the complexity of plant cell wall polysaccharides structure for example a diversity of Xylo glucan sidechain structures has been identified in different plant species and tissues each unique silo glucan sidechain structure is given a unique signature one letter code as shown in the table two other classes of hemi cellulose –is are the mixed linkage glucans and the man ends the mixed linkage glucans which are abundant in grasses and equi sedum stand out as the only class of Hemi cellulose is that do not have a strict beta 1-4 linked backbone rather these polysaccharides consist predominantly of stretches of three or four beta 1-4 linked glucose units adjacent to single beta one three linkages yielding a kink in the polymeric structure the man ants are family of polysaccharides that contain beta 1-4 linked man and/or glucomannan backbones in the glucomannan z’ single glucocil residues are interspersed within the mane and backbone the man ends and glucomannan may be branched with alpha one six linked galactose and the backbone of glucomannan may be acetylated at o2 and or o3 alack domains are particularly abundant and seed storage tissues particularly in legumes while the glucomannan are abundant in gymnosperm secondary walls the final family of hemi cellulose is silent grass primary walls and all higher plants secondary walls have large amounts of the hemi cellulose xylem dicot primary walls have lesser yet significant amounts of xylan the specific decorations of the xylem backbone vary depending upon the species and tissue type all’s Islands have a backbone of beta 1-4 link silos thus like cellulose silo glucan and the man ends the glycosyl residues in the xylem backbone flipped 180 degrees with every other residue leading to a ribbon-like structure however the frequency and type of branching of the backbone as well as the three-dimensional orientation of those branches affects the portion of xylan available for hydrogen bonding to itself or to cellulose or other Hemi cellulose –is xylan and hardwood as depicted in the top panel is branched with alpha glue guy or Forel mouth or bouquet at the o2 position of approximately every eighth xylose residue in the backbone the frequency of glue k2 for O methyl glue k is roughly 1 2 3 in softwoods as shown in the middle left panel the xylan is more frequently glucuronidation with methyl glue k residues at roughly every 5th to 6th xylose in the backbone softwoods island is also more heavily Arab and oscillated than in hardwoods Woods Island is often acetylated at o2 and o3 an oligosaccharide sequence of ram nose galley and xylose as shown in the middle panel has been identified at the reducing end of xylan that has been

characterized from dicots and gymnosperms however there is no evidence for this reducing end oligosaccharide sequence in grass silent brass island as shown in the bottom right panel is much more heavily alpha Arab and oscillated at o2 and o3 than in wood and is also less frequently glucuronidation in addition some of the arabinose in grass silane contains very late esters @o v which may serve as primers for lignin fashion the fur relates may also dimerize thereby cross-linking the cylon in the wall cereal grains Island does not contain glucuronic acid and is referred to as a ravenous island we now move our attention to have my cellulose synthesis analyses of expressed sequence tags libraries and other genes sequence databases revealed the cellulose synthase superfamily which includes both the sesay genes and the cellulose synthase like CSL gene families all these genes belong to the glycosyltransferase to gt2 family in the kz database multiple lines of evidence indicate that the backbone of the hemi cellulose as’ are synthesized by members of the different CSL plates as depicted in the phylogeny diagram it is important to note that the CSL F H and J clades are specific to the grasses to date man an and glucomannan synthesis have been identified in the CSL a clade and man and synthesis have also been identified in the CSL D clade Luke Hanson thesis involved in mixed linkage glucan synthesis have been shown to reside in the CSL F and aged clades the available evidence supports the CSL C clade as being involved in Xylo glucan backbone synthesis several CSL proteins have been localized to the golgi consistent with the location of synthesis of the Hemi cellulose is in the Golgi interestingly a topology study of CSL a and CSL see family members has shown that CSL a9 am an ant synthase has an odd number of transmembrane domains and an active site facing the lumen of the Golgi while CSL c4 acclaimed silo glucan glucan synthase has an even number of transmembrane domains and its active site faces the cytosol thus CSL c4 has a topology similar to SAS a xylem glucan synthesis has been extensively studied and the genes encoding the glycosyltransferase –is that synthesized the backbone and common Arabidopsis branch structures have been identified here as in all our slides depicting all synthesis the enzymes which have been enzymatically confirmed via heterologous expression studies are noted with an Asterix the enzymes that synthesize the glucan backbone as well as those that add the alpha xylose beta galactose and alpha fucose to produce the typical xyla glucan structure found in Arabidopsis arial tissues have been identified as of two putative Xylo glucans specific o acetyl transferases it is important to note that these branching and modifying enzymes unlike CSL c-4 have single transmembrane domains and a so-called type 2 membrane topology with their catalytic sites in the lumen of the golgi several lines of evidence including co-expression and protein-protein interaction studies indicate at the backbone synthesizing enzyme CSL c4 and several of the X X T’s may function together in one or more protein complexes based on these data a model has been proposed as shown on the right in the figure CSL c4 is depicted as synthesizing a glucan chain on the cytosolic side of the Golgi membrane and the nascent glucan chain passes through the membrane into the Golgi lumen where the X X T’s begin the process of sidechain synthesis by zile oscillation of the glucan chain the fact that mixed linkage glucans are not found in dicots presented a unique system to confirm enzyme activity of putative mixed linkage glucan biosynthetic enzymes that were identified via comparative genomics and transcript expression analyses during grain development heterologous expression in dicots such as a rabid abscess and tobacco of rice CSL F genes and a member of the barley CSL h clade resulted in the production of mixed linkage glucans in these dicot systems the data indicate that both the CSL f and c SL h enzymes are able to

synthesize both the beta 1-4 and beta 1 3 linkages in the mixed linkage glucans mechanistic how this process occurs is still under study twelve members of the CSL a family from different species have been shown to have man and synthase activity through heterologous expression in Drosophila and Pakeha eight of these also have glucomannan synthase activity these results clearly show that the multiple members of the CSL a clade catalyzed the synthesis of man an and glucomannan backbones in addition several members of the CSL D clade when transiently expressed in the Koshien a– Bentham Jana leaves yielded increased man and synthase activity suggesting that members of the CSL D clade may also be involved in man and backbone synthesis the Galactus Hill transferase that adds alpha galactose onto the o6 position of the mane and backbone was the first wall biosynthetic enzyme for which its gene was identified the galacto man and Galactus sill transferase has been shown to be responsible for the degree of blacked oscillation of galacto manon’s a property that affects the industrial utility of the Siege storage polysaccharides the definitive identification of the enzymes that synthesize Island has been and remains challenging mutant studies identified a series of a rabid abscess irregular xylem irx mutants from which a subset have been shown to have modified silent structure thereby implicating the affected genes as involved in silent synthesis detailed characterization of the cell walls from such mutants has led to the assignment of putative function for many of the enzymes encoded by these genes as shown in the top panel six different irx genes 9 10 and 14 and their homologs are implicated as involved in zile and backbone synthesis four genes are x-77 like ate and harvest are proposed as putative ly involved in the synthesis of the reducing and a legacy caride sequence the putative acetyl transferase es k1 has been identified enzyme activity has been confirmed for the Arab adopts as methyl transferase GX mt1 and it’s to homologs that methylates the o4 position of glucuronic acid in blue Kira knows Island in addition several of the Guk’s genes have been shown to encode root karana sill transferases that add blue k onto the xylan backbone evidence for the production of unique patterns of glucuronidation of the xylem backbone by the different Guk’s enzymes has also been reported the enzymes that catalyze the synthesis of grass luciano arabinose islands are less well understood over expression of the rice putative orthologs of ir x99 light and 14 complimented the arab adopts acai our X 9 and IR x 14 mutants indicating that the rice genes have a similar function as the Arab adopts as counterparts the heterologous expression of wheat and rice XA T’s in a rabid abscess resulted in 1/3 Arab annihilation of the daikon Island implicating these jeans as xylan Arab initial transferases a putative side-chains Alisal transferase XA x has also been identified we now move our attention to the final family of plant cell wall matrix polysaccharides pectin pectins are a complex family of polysaccharides or polysaccharide domains that are enriched in the middle lamella that junction between two cells correspondingly pectins are known to have roles in cell cell adhesion however the pectin polysaccharides or polysaccharide domains which include REM local actor nan one homo black turnin and Ramla black turn and two are also present throughout the primary wall being most abundant in dicot primary walls but also present in grass primary walls and in secondary walls the pectin polysaccharides interact both non-covalently and covalently with each other a covalent interaction is illustrated by the calcium salt bridges between adjacent negatively charged regions of homo black Ternan but they also interact covalently as shown by the borate diaster cross links between two round no black turn and two regions leading to so-called Bramblett lectern and two dimers in the wall furthermore round neglect nn1 and homo Glac Ternan have

been shown to be covalently linked via their backbones and round neglect turnin to is covalently linked to homo clack turnin finally the homo black turn end can exist in a highly methyl esterified form pectins are also known to interact with cell wall receptors such as walk proteins and as mentioned earlier at least some pectin domains are covalently linked to proteoglycans such as a pop one so what are pectins pectins are defined as cell wall polysaccharides that contain alpha electronic acid or alpha gal a linked at the 1 and 4 position this family of pected polysaccharides include the most abundant pectin oma black Ternan also called hg accounting for 65% of pectin along with the less prevalent Xylo black turn an and AP electornic ram neglect turn in one is the next abundant pectin representing 25% of pectin and is referred to as RG one finally ram neglect turn and two accounts for 10% of pectin and is called RG 2 the most abundant pectin Homa black turn an is also the simplest it is a linear polymer of alpha 1-4 linked khalaq tronic acid 22 more than 80 percent of the galleys in the HG backbone may be methyl esterified at the carboxyl group hg may also be assimilated at o2 and o3 plant walls may contain lesser amounts of the substituted hg zilog lectern n in which some of the gallais residues in the homo black turn and backbone are branched with single beta xylose residues at the old 3 position some aquatic plants THG substituted with single or double apos residues at the o2 or o3 position of gali the most structurally complex of the peptic polysaccharides is Ram neglect turn and to RG 2 RG 2 consists of a homo black turn and backbone to which for complex side chains are attached RG 2 consists of 12 different sugars with over 20 different linkages sidechain a and sidechain B are linked to the homo black turn and backbone via a POS and they are decorated with a series of different sugars in different an American figurations and linkages sidechain C contains the 8 carbon sugar KD o and sidechain D contains the 7 carbon sugar DHA the apos inside chain a is the position at which the borate diaster occurs between 1 RG 2 molecule and that same apos residue in another RG 2 molecule the formation of RG 2 dimers is known to be important since mutants that have even small structural modifications that lead to a reduction in RG 2 dimer formation are dwarf plants suggesting a role of RG 2 dimers in plant growth Ram neglect or nan 1 RG 1 is unique among the pectin polysaccharides in having a disaccharide repeat backbone the backbone of RG 1 consists of alpha galactic acid alpha gal a linked to the 2 position of alpha l ram nose which is then linked to the four position of the next gal a residue resulting in a disaccharide repeat along the entire backbone most or all of the Gally residues are all acetylated at the o2 or o3 positions o asset elation of the rhamnosus has also been reported 25 to 80 percent of the rhamnosus in the backbone are substituted at the 4 position unlike the side chains of RG 2 which have a very conserved structure across all plants the side chains of RG 1 vary depending upon the stage of development and cell type the examples shown in this are the rg1 side chains that have been structurally identified as attached to the for position of the RG one backbone Ram knows residues in general RG one side chains include beta one four linked collectin which may be branched at the O three position with L arabinose or with the rabbin and beta one three linked Galacta a branched at the O six position with Galacta nor rabinal actin an alpha one five linked a rabbin n which may be branched with arabinose or Rabun an at o2 and o3 some RG one side chains may also contain single fucose residues such as shown in the third side change structure in the slide as well as with glue K or with 4o methyl glue Gaye an important question in regards to pectin structure is how are the different pectin polysaccharides connected to each other in the wall to make larger macro molecular structures in this figure we show the different types of

interconnections that have been structurally confirmed between the different pectin domains as mentioned previously are G 1 and HG are connected via their backbones as rh g & RG 2 however whether or not our G 1 h g & RG 2 all exists within a single structure remains uncertain at the present time finally the existence of the proteoglycan a pop one reveals that some RG 1 and HG domains exist covalently attached to a proteoglycan several of the genes that encode the pectin biosynthetic enzymes have been identified and we will review those here the most detailed studies of pectin synthesis have been targeted at understanding the synthesis of homo Galacta an HG this work has led to several surprises the HG backbone is synthesized by alpha 1-4 electron acyl transferase –is or HG gal eighties the Arab adopts is gelt 1 short for galacto an acyl transferase one is the first identified and biochemically confirmed HG gal 80 interestingly as illustrated in the model on this slide got one has been shown to exist in a disulfide bond at protein complex with gout seven which is another member of the Arab adopts Asst 15 member gout gene family interestingly although gout one based on its encoded amino acid sequence is predicted to be a type 2 membrane protein with a single trans membrane spanning domain near the end terminus in Arabidopsis out one apparently undergoes post translational processing in planta resulting in the loss of the transmembrane domain this led to the question of how gout one was retained in the Golgi the site of pectin synthesis without a transmembrane domain it was shown that the truncated gout one is anchored within the Golgi apparatus through its association with gout 7 in the got one gout 7 complex immuno precipitation of the Godwin gout 7 complex coupled with mass spectrometry experiments have identified 12 additional putative interacting proteins but the full mechanism of hamad lectern and synthesis remains to be elucidated as mentioned earlier the h g backbone is modified by methyl astera fication and acetylation 3 putative h g methyl transferases cg r 3 Guate and qua 3 have been identified although enzyme activity from these proteins still needs to be demonstrated several mutants have been reported to have reduced pectin astera fication with the underlying genes TBR t BL 3 and p.m r 5 being members of the TB are related gene family whose other members include the putative Xylo glucan and xylan OS Edel transferases presented earlier in the Hemi cellulose biosynthesis section whether or not t BR TBL 3 and PM r 5 are indeed pectin acetyl transferases that act on hg remains to be determined assuming that a unique enzyme is required to catalyze the formation of each unique pectin glycosidic linkage and modification the complexity of pectin structure indicates that at least 67 different transferases including glycosyltransferase –is methyl transferases and acetyl transferases are required for the synthesis of pectin yet so far only seven genes have been concludes they identified to encode pectin biosynthetic enzymes and they comprised only four different transferase activities illustrating the relatively slow progress in understanding the biosynthesis of this most complex family of wall polysaccharides not one which we discussed in the previous slide is one of the seven genes another five genes are shown here and the last one will be presented in the next slide xgd one encodes a beta one threes a little transferase that adds ILO’s on to the galley residues in the HG backbone to produce silo collector Ihnen in RG two biosynthesis for our GXT genes have been identified that encode alpha one three Zyliss sill transferases which adds ILO’s onto the fukusa residue inside chain a of RG two it is clear that much research is still needed to determine the components of the RG two biosynthesis machinery progress has been made in identifying enzymes involved in the synthesis of the side chains of RG one the arab adopts is g ALS one gene has been shown to encode a beta 1-4 galactus hill transferase that elongates the beta 1-4 galactus ID chains of RG 1 its homologs G ALS 2 and 3 are also proposed as putative beta 1-4 Galactus sill transferases since they’re mutants display a similar reduction in the amount of cell wall galactose as observed in the G ALS one mutant however

their enzymatic activities remain to be demonstrated characterization of a rabid OPS’s T DNA insertion mutants also implicated air at one and its homolog era two in the biosynthesis of the Arab anon side chains of RG one identifying them as putative alpha 1 v arabinose LLL transfer races however their enzymatic activities remain to be demonstrated recently a protein-protein interaction study employing techniques including by molecular fluorescent complementation förster resonance energy transfer and non reducing SDS page indicated that arid one and arid to form homo and hetero that contain disulfide bonds these data suggest that enzyme complexes may be involved in the synthesis of a rabbin and side chains of rg1 the enzymes and the encoding genes that synthesize the rg1 rhamnosus gal a disaccharide repeat backbone are not presently known it is important to stress as we conclude this presentation that although the major cell wall polysaccharides have been identified and characterized we still do not understand the fine structural details of the wall how the polymers interact or how the biosynthetic enzymes work together to produce the plant cell wall indeed research is needed to understand cell wall architecture and its synthesis for example the recently discovered plant cell wall proteoglycan a pop one provides one example of how the generally considered separate wall matrix polysaccharides pectin and xylem are connected in one covalently linked structure whether additional wall proteoglycans exist and the prevalence of a pop one in plant cell walls our topics of current investigation but the fact that such new wall structures exist and are being identified make evident that much remains to be discovered about the plant cell wall its structure and its biosynthesis an increased understanding of the architecture of the plant cell wall and the enzymes that produce it is necessary to make the best use of this critical renewable resource as we move into a future with a growing human population and an increasing need for a sustainable economy the summary of plant cell wall structure and synthesis represented in this talk is based on the work of many many investigators we’ve identified many of the papers that support this presentation throughout the slides but we add here additional references we wish to also acknowledge Stephan Everhart for much of the photography on these slides and the funding agencies that have supported the research over the years on the plant cell wall and in particular that have supported our work the US Department of Agriculture than National Science Foundation in the US Department of Energy