Plant PolysaccharidesBCMB 8020- overview of plant glycobiology- plant cell walls- types of plant cell wall polysaccharides: structure and biosynthesis- RG-II: a curious case in plant glycobiology- special functions of plant polysaccharides- biofuel- green plants constitute about half of the living matter on earth- plants synthesize many of the same types of oligosaccharides that are found in animals but also produce a wide variety of unique sugar chains- many of the earliest studies on carbohydrates were done in plantsGeneral Organization and Features of a Plant Cellvacuole = lysosome/storage granulesymplast = shared cytoplasmplant cells were first described by Robert Hooke who noted that slices of cork looked like “cells” in a monastery under his early microscopePlants form two types of cell walls: a primary cell walls that differ in composition and function:Primary cell wall: highly flexible, found in growing plant cells, polysaccharide-rich (80-90% polysaccharide; 10-20% protein)Secondary cell wall: thicker and stronger, contains majority of plant biomass, lignin, a macromolecule of cross-linked phenolic structures, is a major component of the secondary wall, polysaccharides are also present but in relatively lower proportion and altered contentPrimary cell wall is the extracellular matrix of plants- contains many types of polysaccharides; main polysaccharides are:cellulosehemicellulosepectins- plant cells also contain several structural glycoproteins that are rich in hydroxyprolines and heavily modified by serine and threonine- linked oligosaccharides (O-linked)extensinsproline/hydroxyproline-rich glycoproteinsarabinogalactansCellulose: structure and biosynthesis- cellulose is a long, linear polysaccharide consisting of ß1,4 linked glucose residues; the most abundant biopolymer in nature- forms a crystalline microfiber via extensive hydrogen bonding between individual cellulose polysaccharides- biosynthesis of cellulose occurs at the plasma membrane using cytoplasmic UDP-glucose generated from sucrose- cellulose provides tensile strength to plant cell matrix (similar to role of collagen fibers in animal cells)Cellulose biosynthesis involves nearly 40 different CESA proteins at the plasma membrane and is functionally tied to microtubulesrosetteHemicellulose: structure and biosynthesis- hemicellulose consists of ß1,4 linked glucose residues (like cellulose) that are substituted with other sugars; xyloglucan is the predominant hemicellulose - others include glucuronoxylan, arabinoxylan, glucomannan, and galactomannan- coat and cross-link cellulose fibers in the primary cell wall- as cells increase in volume, H-bonds that link cellulose and hemicellulose loosen, allowing the internal osmotic pressure of the cell to push apart the cellulose microfibers; this process is critical for plant cell growth and is helped by enzymatic cleavage of xyloglucan chainsPectins: structure and biosynthesis- pectins are complex acidic polysaccharides that contain 1,4-linked !-D-galactosyluronic acid residues; they resemble glycosaminoglycan (GAG) chains- pectins that have been characterized include homogalacturonan (65% of plant pectins), substituted galacturonans like apiogalacturonan and xylogalacturonan and rhamnogalacturonan I and II- embedded within the cellulose/hemicellulose network, pectins provide hydration and additional strength to the primary wall- pectins can be modified by methyl esterases; extent of methylation can determine porosity and stiffness of cell wallDetermination of pectic polysaccharide structure requires the fullrange of available analytical techniquesRhamnogalacturonan I (RG-I): structure and biosynthesis RG-I consists of a 4-!-D-GalpA-(1,2)-!-L-Rhap backboneGalA residues are not usually substituted in RG-IRhamnogalacturonan II (RG-II): structure and biosynthesis RG-II consists of a 1,4-!-D-GalpA backbone- RG-II is substituted with four highly complex and conserved side chains that contain 12 different sugars in more than 20 different linkages8922RG-II is a major polysaccharide component of red wine- 1 liter of red wine may contain between 100 and 150mg of RG-II (white wine typically contains 20 - 30 mg of RG-II per liter) - differences result form the different processing used to make these wines- white wines are made by fermenting grape juice which contain little of the cell wall but red wines are made by fermenting grape berry pulp- the cell wall accounts for a large portion of the pulp and the RG-II is solubilized during the fermentation process- RG-II is extremely resistant to known microbial polysaccharide degrading enzymes and thus is not utilized as a carbon source during fermentationWhy do higher plants invest so much energy and effort in producing such a complicated structure in their wall?- first clue came from observations that RG-II exists as boron- cross-linked dimer (between two apiose residues on the side chain A of an RG-II monomer)two stereoisomers of borate-linked RG-IIsFormation of the borate ester cross linkBor1 mutant: mutation in a boron transporter- WT and bor1 RG-II have similar glycosyl residue compositions but bor1 has much lower levels of cross-linked monomers- boron (and other metals) are important components of plant cell wallsand play a role in cross-linking pectin- does this prove that RG-II is also important for cell growth?Changes in boron content affect plant growthDoes the absence of RG-II dimers cause growth defects?- to address this question, different mutants in Arabidopsis were studied including the pleiotropic mutant, mur1- mur1 mutant identified by screening hydrolysates of chemically modified plants; characterized by fragile cell walls and dwarfed size- found to be deficient in GDP-mannose-4,6-dehydrase (enzyme responsible for GDP-fucose synthesis)Problem: fucose is not only found in RG-II (xyloglucans and glycan side chains of many proteins contain fucose)Which altered polysaccharides are responsible for the growth defect in the mur1 mutant?- another mutant cgl1entirely lacks fucose in its glycoproteins butsurprisingly does not show any phenotypes- mur2 mutants synthesize xyloglucans with only 2% of normal fucoseBut also have no growth defect- effects on RG-II in mur1?Arabidopsis mutants with altered RG-II structure (cont.)- L-galactose is substituted in place of fucose; this leads to reduced amounts of RG-II dimer, less stable dimers and a slower rate of