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The Plant Cell Wall Why have a cell wall? • Plants have ‘discovered’ the ecological niche of photoautotrophy. • To successfully compete for sunlight, plants need to grow (high, fast, directed, adaptive). • To support growth, plants had to ‘invent’ construction material that was dynamic and mechanically stable. • The cell wall is a complex molecular network of carbohydrates and glycoproteins that is both. Plant cell shape is defined by the cell wall root growth movie Plant cell shape is defined by the cell wall Every plant cell is surrounded by a wall primary cell wall (mechanical stress): cellulose, hemicellulose, glycoproteins primary cell wall (cell adhesion and separation): pectin, soluble glycoproteins, enzymes, peptide 1mm The cell wall is involved in every aspect of plant life division defence expansion secondary wall thickening morphogenesis Cell walls and plant health • The cell wall is the first line of defence against pathogens • The cell wall is actively degraded by many pathogens • An infected plant locally modifies its cell wall to defend itself • Cell walls are modified or newly made during parasitic and symbiotic plant microbe interactions (e.g. nematode syncytia, arbuscular mycorrhizal plant cell interface) • Genetic alterations of cell wall polymers can lead to systemic pathogen resistance and enhanced tolerance of abiotic stress (drought, cold etc.) Cell wall molecular biology • Structure • Biosynthesis (biochemical and genetic perspective) • Map based cloning of biosynthetic genes • Cell biology or oriented cell wall deposition • Cell wall remodelling/loosening • Signal function of cell wall carbohydrates Primary cell walls consist of interacting networks carbohydrates Cell wall fractions Cellulose insoluble in aqueous buffers, Glc Cell wall matrix polymers Hemicellulose neutral, soluble in alkali, Glc, Xyl, Gal, L-Fuc, GlcA Pectin acidic, soluble with Ca2+-chelating buffers, GalA, L-Rhm, L-Ara, Gal Glycoproteins partially water soluble, lipid-anchored or bound to other polymers, Gal, L-Ara L-Rhm, GlcA Enzymes mostly soluble Lignin insoluble, only secondary walls, polyphenolic Heterogeneity of cell wall components Plant cell wall carbohydrates contains ca. sixteen different monosaccharides also present: Aceric acid, L-galactose, N-acetyl-D-glucosamine, Kdo, Dha Carbohydrates consist of chains of sugars linked in a specific way Cellulose is a linear polymer (1→4)β-D-glucan Dozens of cellulose chains form paracrystalline microfibrils Xyloglucan is the main hemicellulose in dicot primary cell walls (1→4)β-D-glucan backbone with (1→6)α-D-xylose side chains Glucuronoarabinoxylan is the main hemicellulose in dicot secondary cell walls (1→4)β-D-xylan backbone with (1→2)α-D-glucuronic acid and (1→2)α- Larabinose side chains Pectin consists of three different polymers Homogalacturonan (HGA) methylesterified (1→4)α-D-galacturonan Developmental variation of HGA esterification highly esterified relatively de-esterified Rhamnogalacturonan I (RG I) contains different side chains (1→5)α-L-arabinose (1→4)α-D-galactose →2)α-D-rhamnose-(1→4)α-D-galacturonan Rhamnogalacturonan II (RG II) has the most complex known carbohydrate structure Cell wall proteins contain hydroxyproline • Ara modification is predicted by the Ser(Pro)4 sequence • Extensins forms stiff rods. • Extensins can be oxidatively cross-linked by the action of cell wall peroxidases. • Extensin cross-linking might rigidify the cell wall after expansion has seized. Arabinogalactan-proteins (AGPs) contain lipid anchors and very complex carbohydrate modifications Arabinogalactan-proteins (AGPs) • AG-modification predicted by Pro-Ala-Pro-Ala sequence. • hydrophobic C-terminus is replaced by cleavable glycolipid (GPI-anchor). • hundreds of different AGP like proteins exist in the cell wall. • AGPs have been implicated with many biological roles. Summary cell wall structure/components Cellulose Cell wall matrix polymers Hemicellulose xyloglucan (XG) glucurono(arabino)xylan Pectin homogalacturonan (HGA) rhamnogalacturonan I (RGI) rhamnogalacturonan II (RG II) Glycoproteins extensin arabinogalactan-proteins (AGPs) Enzymes glycosyl hydrolases esterases peroxidases ... The biosynthesis of complex carbohydrates depends on the supply of activated monosaccharides free sugar activation nucleotide sugar Interconversion generates new sugars glycosyl transferase polymers and conjugates NDP-sugars are interconverted by oxidoreductases and isomerases UDP-D-Glc -4-epimerase UDP-D-Glc UDP-D-Gal NAD+ UGD NADH UDP-D-GlcA -4-epimerase UDP-D-GalA UDP-D-GlcA UXS CO2 UDP-D-Xyl -4-epimerase UDP-D-Xyl UDP-L-Ara Biosynthesis takes place at different cellular locations How can the molecular machinery of cell wall biosynthesis be determined? • biochemical approach • forward genetic approach • heterologous expression of candidate genes • systems biological approach Biochemical-molecular dissection of cell wall biosynthesis • • • • • • • Reconstitute reaction in cell-free system Acceptor and donor substrate Detect product Solubilize (!) and purify the activity Isolate the enzyme Sequence the peptide(s) Clone the cDNA, gene Example: Cloning of β-mannan synthase • In some plants hemicellulose acts as storage polysaccharide analogous to starch • guar, fenugreek, locust beans, coconut: galactomannan • tamarind: xyloglucan • galactomannan is used as food stabilizer (e.g. ice cream) Example: Cloning of β-mannan synthase • Reaction was assayed in microsomes of developing guar seeds by detecting radioactive β-mannan using endomannanase • The enzyme was solubilized using digitonin • Peptides of a relatively pure fraction were partially sequenced • A new library of 15000 cDNA clones of the source tissue was made and sequenced • The matching clones were assembled to obtain a full sequence • The gene was functionally expressed in soybean cells • The mannan-synthase belongs to the family of cellulose synthase-like genes (Csl). • The work was done at Pioneer Dupont (Dhugga et al 2004 Science 303: 363ff) Further examples of biochemical cloning • xyloglucan-specific fucosyl transferase (FUT1) • galactomannan-specific galactosyl transferase • homogalacturonan (HG)-specific galacturonosyl transferase (GAUT) Molecular genetic dissection of cell wall biosynthesis • Devise/perform a screen (based on hypothesis) • Characterize the mutant • Clone the gene Example: Cloning of REB1, a gene required for normal roots and arabinogalactan-protein (AGP) composition 2D electrophoresis of root AGP REB1 (WT) reb1 reb1 is a recessive single locus X P ↓ F1 ↓ F2: 75%:25% Single loci can be identified by mapping ecotype L X ecotype C ↓ F1 ↓ F2: 75%:25% Single loci can be identified by mapping { : ≈ 50% : ⇒ 100% markers Markers can detect differences in DNA sequencel/length at a defined chromosomal locus (e.g. PCR fragments, restriction sites, single nucleotide polymorphisms SNPs...) For fine mapping, plants containing recombinations close to the mutant locus are selected • DNA polymorphisms close to the locus have to be used / identified. • Thousands of mutant F2 plants are screened to find sufficiently close recombinants. Gene identification by direct sequencing, KO allele selection and complementation. • Gene annotation databases for Arabidopsis: http://atidb.org/ etc. • Mutant collections: SALK, SAIL .... • gDNA Clone collections: ABRC ... • general Arabidopsis portal: TAIR http://www.arabidopsis.org/ • Before the Arabidopsis genome was completely sequenced the work took two years of one postdoc. (e.g. Seifert et al. 2002 Current Biol. 12:1840ff). • Nowadays map based cloning takes one MSc student 3 to 6 months and is offered commercially. ATIdb REB1 encodes an enzyme required for UDPGal biosynthesis AGP UDP-D-Glc -4-epimerase UDP-D-Glc UDP-D-Gal pectin NAD+ UGD hemicell. NADH UDP-D-GlcA -4-epimerase UDP-D-GalA UDP-D-GlcA UXS CO2 UDP-D-Xyl -4-epimerase UDP-D-Xyl UDP-L-Ara Biochemical-molecular dissection of cell wall biosynthesis. Pros and cons? • • • • • • Advantages access to mechanism addition of cofactors etc. direct response kinetic observation you know what you get • Disadvantages • obtaining substrates • setting up the assay conditions • identifying source containing high activity • availability of material • instability of isolated enzyme • sometimes no cDNA library available • system disrupted • artificial conditions • no in vivo function Molecular genetic dissection of cell wall biosynthesis pros and cons? • Advantages • many screens possible • system intact • in vivo function • map-based cloning is straightforward • Because of phenotype the functionality of modified versions can be assayed in vivo. • epistasis analysis • multiple KO mutants • Disadvantages • some screens are cumbersome • no mechanism • abiotic stress • compensation • genetic redundancy • lethality • you don t know what you will end up with Genetic screens that have elucidated cell wall biosynthesis/function • root morphology • xylem morphology • cell wall carbohydrate composition Some root morphology mutants • Genes required for primary cell wall (growing) • root swelling • root hair deficient • things falling apart • salt overly sensitive • cobra, procuste, korrigan, quasimodo, kojak ... xylem/fibre morphology mutants • Genes required for thickening cell wall (secondary) • irregular xylem: IRX • fragile fibre: FRA Most CesA genes were isolated by Arabidopsis genetics Three CesA genes form the catalytic core of plant cellulose synthase Primary cell walls: CesA1, -3, -6 Secondary cell walls: CesA4, -7, -8 Two different sets CesA isoforms are necessary for primary and for secondary cell wall formation CesA multimers might form ‘rosette’ strucutres at the the plasma membrane Several Arabidopsis genes involved in cellulose synthesis are unclear in their function • • • • • KORRIGAN (3 genes): membrane bound β-1-4 glucanase COBRA (11 genes): GPI-anchored, novel KOBITO (3 genes): PM-localized, novel POM POM1: endochitinase (?) TBR1 (several genes): PM-localized, novel • The difficulty to annotate biochemical functions to these gene products highlights the limitation of forward genetics. • It is doubtful whether the novel genes would have been found otherwise. • Forward genetics has the potential of discovering not only new genes but also new processes. Carbohydrate compositional mutants: The MUR genes • collect leaves • extract proteins lipids etc. → crude cell walls • hydrolyze poly- to monosaccharides • derivatize for gas chromatography (GC) • quantify on GC + mass spectroscopy (MS) • 11 mutant loci identified from 5000 mutagenized plants • 6 cloned • nucleotide sugar metabolism (2) • glycosyl transferases (3) • novel (1) Systems biology approach to functional gene identification • Systems biology is the study of the interactions between the components of a biological system, and how these interactions give rise to the function and behaviour of that system (for example, the enzymes and metabolites in a metabolic pathway)." Systems biology approach to functional gene identification • • • • • • Systems biology is the study of the interactions between the components of a biological system, and how these interactions give rise to the function and behaviour of that system (for example, the enzymes and metabolites in a metabolic pathway)." Example: transcriptional co-regulation of a metabolic pathway dedicated to secondary cell wall formation." Relative transcript abundance for >22000 Arabidopsis genes has been analysed in hundreds of different stress and developmental conditions, tissues, cell types etc." Pairwise comparison of transcript abundance identifies co-regulated genes." Transcriptional co-regulation is a hint to involvement in the same biological process." This hypothesis can be tested by reverse genetics using a large collection of TDNA or transposon tagged Arabidopsis mutants, or by gene silencing." Example: Genes co-regulated with secondary cell wall specific CesA genes Primary cell walls: CesA1, -3, -6 Secondary cell walls: CesA4, -7, -8 Two different sets CesA isoforms are necessary for primary and for secondary cell wall formation Pairwise comparison http://affymetrix.arabidopsis.info/narrays/twogenescatter.pl • RSW1 = CesA1 (primary walls) • PRC1 = CesA6 (primary walls) • IRX3 = CesA7 (secondary walls) • IRX5 = CesA4 (secondary walls) CSB.DB: Comprehensive systems biology database IRX7 co-regulated genes Mutant Phenotype IRX6 IRX5 IRX8 IRX12 IRX1 No IRX9 nd nd nd No nd No nd nd No IRX ? The molecular biology of cell wall biosynthesis today • Most nucleotide sugar interconversion enzymes have been cloned. • Cellulose synthase requires three isoforms of CesA genes. • Many genes that are essential for cellulose biosynthesis (e.g. COBRA, KORRIGAN, POMPOM) are presently lacking a clearly defined biochemical function. • β-mannan synthase is encoded by CslA genes. • Xyloglucan fucose and galactose side chains are attached by the MUR2 and MUR3, glycosyltransferases. • Several ‘hot candidate genes’ for a functioning in matrix polymer synthesis from forward and reverse genetics and biochemistry. • There are still huge gaps in our knowledge. • The database for carbohydrate active enzymes (CAZY) contains a disproportionate number of plant genes. Most of unknown biochemical and biological function. Cellulose microfibrils (CMF) are aligned perpendicular to the growth direction Cell wall deposition during elongation growth is highly anisotropic Sugimoto ea. 2000 Anisotropy of load-bearing cell wall polymers is a precondition of anisotropic expansion turgor pressure during growth: 10 bar tensile strain on 0.1µm thick primary wall: 5000 bar Two molecular problems of anisotropic expansion: • How is anisotropic deposition controlled/achieved? • How can mechanically stable polymers yield to turgor pressure in a controlled manner? ften also microtubules are seen aligned perpendicular to the growth direction Do microtubules control the oriented movement of cellulose synthase? Two molecular problems of anisotropic expansion: • How is anisotropic deposition controlled/acheived? • How can mechanically stable polymers yield to turgor pressure in a controlled manner? How is cell wall loosening controlled? How is cell wall loosening controlled? • XET: xyloglucan endo-transglycosylases • expansin: topoisomerase (?) • (1→4)β-glucanases • non-enzymatic loosening by hydroxyl radicals (OH•). Enzymes involved in fruit softening • pectin hydrolases • pectate lyases • pectin esterases • expansin • glucanases Cell separation is locally controlled • The action of pectin hydrolases, - lyases and esterases has to be restricted to small area at the cell corners to allow tissue growth but prevent that things are falling apart. Control of cell wall anisotropy • Morphogenesis depends on spatial anisotropy of load bearing cell wall polymers esp. cellulose. • cellulose microfibrils are aligned perpendicular to the growth axis. • Oriented movements of cellulose synthase partially depend on microtubules but the are additional mechanisms. • Controlled cell wall creep is mediated by enzymes acting on the cross-linking glucans (e.g. expansin, XET). • The important problem of local cell separation is elusive