Plant Cell Wall Structure and Biosynthesis Prof. Debra

Plant Cell Wall Structure and Biosynthesis

Prof. Debra Mohnen & Dr. Melani A. Atmodjo

1The screen versions of these slides have full details of copyright and acknowledgements

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Prof. Debra Mohnen & Dr. Melani A. AtmodjoComplex Carbohydrate Research Center

The University of GeorgiaAthens – Georgia

Photography by Stefan Eberhard

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~1/3 is produced in marine plants and microorganisms and ~2/3 is from land plants

Plant cell walls constitute the bulk of plant biomass: ~4-20% plant fresh weight

From Arabidopsis leaf, silique, stem, inflorescence cell walls Caffall, Ph.D. dissertation, 2008, The University of Georgia

CO2 O2

H2O

Each year ~1011 tons of CO2are fixed via photosynthesis into biomass

3

Plant cell walls: cell type-specific extracellular matrices

that surround the >14 types of plant cells

Arabidopsis sepal epidermis

Arabidopsis trichome Arabidopsis pollen

Arabidopsis petal epidermisPoplar stem cross section

Tobacco suspension culture cells




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Plant cell walls have many functions in the plant

• Structure

• Growth

• Flexibility

• Hydration

• Defense

• Signaling

• Cell:Cell adhesion

• Development

From: Persson et al., 2007, Plant Cell 19: 237

WT

gaut12 mutants

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Plant cell walls have many usesfor humans and animals

• Clothing

• Wood & lumber products

• Biomaterials:

– Nanocellulose / nanocomposites

– Biofuels

– Chemicals

• Animal food

• Human food and fiber

• Gelling and stabilizing agents

• Nutraceuticals/pharmaceuticals

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Two general types of walls:primary vs. secondary

Cross section of Nelumbo nucifera petiole showing primary cell wall

(From: Esau, 1977, Anatomy of Seed Plants)

Plant cell walls

Type II (Grasses)

Type I (Dicots)

Two types of primary wall:

Tilia stem cross section

Arabidopsis Switchgrass

Arabidopsis (a) seedlings, (b) callus, (c) suspension culture

80-90% carbohydrate, ~ 10% protein, and in some cell types lignin; wall fine structure is plant, tissue and cell-specific




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Primary walls

• First wall laid down

• Surrounds meristematic (dividing) and growing cells

• Cells in succulent tissues

• Found at the junction of cells and at the outer edges of secondary walls

• Composed of ~90% carbohydrate and ~10% protein

• Two types of primary wall: Type I and Type II

Secondary walls

• Surround cells that differentiate to form specialized functions (i.e. wood, xylem and fibers cells)

• Have altered polysaccharide composition

• Often are lignified

Characteristics of primary & secondary walls

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From: Albersheim et al., 2011, Plant Cell Walls, New York:

Garland Sci.

Type I (dicot) primary wallArabidopsis, electron micrograph

Type II (grass) primary wallMaize, electron micrograph

Plant cell walls consist of interacting polymers

Most plant cell wall models depict three sets of independent

polysaccharide matrices

From: Alberts et al., 2002, Molecular Biology of the Cell 4th ed.

Cellulose

Pectin

Hemicellulose

50 nm

Middle lamela

Primary cell wall

Plasma membrane

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Polymer Cell Wall (mass %)Primary (dicots)a Secondary

Angiosperms GymnospermsCellulose 20-30 37-57 38-52

Lignin 0 17-30 26-36Pectin 30-35 <10 <10

Hemicellulose 25-30 20-37 16-27

a Grass walls contain 2-10% pectin

From: Albersheim, Darvill, Roberts, Sederoff, Staehelin, 2011, Plant Cell Walls, New York: Garland Sci.

• Type I primary walls are abundant in pectin and have no lignin

• Type II walls have less pectin than Type I walls

• Most secondary walls contain lignin and less pectin




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Multiple models of the plant cell wall have been put forward over the past 40 years

However, it is important to note that the lack of complete cell wall structure data means that all current cell wall models are hypothetical,

with some parts of the models being more data-based than others

In their book “Plant Cell Walls”, Albersheim, Darvill, Roberts, Sederoff and Staehelin

chose not to present a single integrated model of the plant cell wallAlbersheim P, Darvill A, Roberts K, Sederoff R, Staehelin A. 2011; Plant Cell Walls. New York: Garland Sci.

Here, we take the same general strategy; however, to provide a conceptual framework we first show a general schematic models

of the wall and then two cell wall models that depict Type I and Type II primary cell walls with emphasis on wall polysaccharides

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The generally accepted plant cell wall model is based on a cellulose-matrix polysaccharide network

From: Albersheim et al., 2011, Plant Cell Walls, New York: Garland Sci.

• Basic cell wall structure:All cell walls consist of a cellulose-matrix polysaccharide network that resists tension (often attributed to cellulose-hemicellulose network) and resists compression and shearing forces (often attributed to pectin network); the pectin rich-middle lamella is the adhesive layer between cells

Cellulose

12From: Carpita & Gibeaut, 1993, Plant J. 3: 1

Cellulose

Pectin

Hemicellulose

Most plant cell wall models represent cellulose, hemicellulose and pectin

(Note: protein is not represented)Type I primary wall Type II primary wall

XyloglucanPGA Junction zone

RG I with arabinogalactan side-chains

GAX

RG I with arabinogalactan side-chains

Xyloglucan PGA Junction zone

Phenolic cross-links




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But what about the cell wall proteins?

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Cell wall proteome

Albenne, Canut & Jamet, 2013, Front. Plant Sci. 4:111; Showalter et al., 2010, Plant Physiol. 153:485

• Cell wall proteins (CWPs) constitute 5-10% of cell wall mass

• Most CWPs are basic proteins

• Most CWPs are post-translationally modified by one or more of the following:

– Hydroxylation of proline

– N-glycosylation

– O-glycosylation

– GPI anchor

• In Arabidopsis, for example, there are 166 types of wall hydroxyproline-rich glycoproteins (HRGPs) including:

– 85 arabinogalactan proteins (AGPs), (highly glycosylated)

– 59 extensins (EXTs), (moderately glycosylated)

– 8 proline-rich proteins (lightly glycosylated)

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Arabinogalactan protein (AGP) distribution in the cell wall

There is great heterogeneity in AGPs:in the protein cores, glycosylation, and cell type expression





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Proposed extensin 3 networks in cell walls

• Extensins have been defined as self-assembling amphiphiles that generate scaffolding networks

• Extensins can be induced in walls by wounding or stress to provide mechanical protection


Lamport et al., 2011, Plant Physiol. 156: 11

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New results show that proteoglycans also exist in cell walls;

How prevalent and diverse they are is just now being investigated

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APAP1 (arabinoxylan-pectin-arabinogalactan protein 1), a proteoglycan containing an AGP core and covalently attached pectin

and hemicellulose, was recently identified in Type I primary wallsTan et al., 2013, Plant Cell 25: 270

Proteoglycans in the plant cell wall

Proteoglycans: proteins that are highly glycosylated

AGP

AG

Xylan1

Xylan2

RG HG RG

Hemicellulose

Pectin

APAP1




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• How ubiquitous is APAP1?

• Do other proteoglycans exist in plant walls?

• Is APAP1 synthesized intracellularly or extracellularly?

Pectin

Hemicellulose

Cellulose

AGP protein

core

Model of cell wall containing APAP1

Based on Tan et al., 2013, Plant Cell 25: 270

The existence of APAP1 demonstrates that our current understanding of wall structure is incomplete

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• Relevant Cell Wall Model

• Representative Structure

• Biosynthetic proteins and process

Structure and synthesis of wall polymers

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Mohnen, Bar-Peled, & Somerville (2008)

In Biomass Recalcitrance –Deconstructing the Plant

Cell Wall for Bioenergy; ed. Himmel, M.E., Blackwell

Publishing, Oxford;Chapter 5: 94-187

Secondary Walls

Some cells with structural roles

CelluloseHemicellulose

↓ PectinLignin

(proteins)

Primary WallDividing and growing cells

CelluloseHemicellulose

Pectin (proteins)

Overview of plant cell wall

biosynthesis

Middle lamella

Nucleotide sugar formation/interconversion

XDP XDP

XDP

Proteins

Pectin Hemicellulose Glycoprotein

Wall-modifying enzymes/proteins

Cellulose synthase

Cellulose

Middle lamella

Pectin

Protein

HemicelluloseXDP

XDPXDPXDP

GTs

O-MeO-AcO-Phe

Courtesy of Malcolm O’Neill

Cellulose

Protein

Lignin

Primary wall

Hemicellulose

Cellulose synthase

Hemicellulose

Proteins

Peroxidase Laccase

Monolignol formation

Transport to wallOxidation

PolymerizationNucleotide sugar

formation/interconversion

Synthesis modification




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• Structural component in many plant secondary cell walls including fibers and water-conducting cells

• Adaptation allowing early vascular plants to colonize terrestrial environments (~500 million years ago); likely a structural and water-transporting role

• Aromatic macromolecular heteropolymer containing p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) subunits

• Produced by combinatorial free radical coupling of the phenylalanine-derived monolignols p-coumaryl, coniferyl and sinapyl alcohol

• One of most abundant organic polymers on earth; Second to cellulose

Lignin

Mansfield et al., 2012, Nature Protocols 7:1579; Bonnawitz & Chapple, 2013, Curr. Opin. Biotechnol. 24:336

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Visualized by (A) autofluorescence, (B) phloroglucinol, and (C) Mäule staining

From: Wang et al., 2010, PNAS 107: 22338-43

Lignin in xylem of Medicago truncatula

A

B

C

24Chemical structure of monolignols and representation of wood tracheid from a coniferFrom: Davin & Lewis, 2005, Curr. Opin. Biotechnol. 16: 407

Three types of monolignols are the main structural units H, G and S lignin

H G S




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Lignin is formed in the cell wall by oxidative polymerization of p-coumaryl, coniferyl, and sinapyl alcohol to yield p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin

From: Hao, Ph.D. dissertation, 2012, The University of Georgia, as modified from Dixon et al., 2001Phytochem. 57: 1069; Humphreys and Chapple, 2002, Curr. Opin. Plant Biol. 5: 224Li et al., 2010, Plant Cell 22: 1620; Zhao and Dixon, 2011, Trends Plant Sci. 16: 227

Major ether and C-C linkages in lignin

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• H and G lignin: deposited during early lignification in middle lamella and cell junctions

• G lignin: deposited earlier in vessels and fibers than S lignin

• S lignin: deposited mainly in fibers

H, G and S lignin are deposited in a spatial and time-specific fashion

Donaldson, 2001, Phytochemistry57: 859

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Arabidopsis general phenylpropanoidpathway for production of lignin precursors

From: Hao, Ph.D. dissertation, 2012, The University of Georgia, as modified from Dixon et al., 2001, Phytochem. 57: 1069; Humphreys and Chapple, 2002, Curr. Opin. Plant Biol. 5: 224; Li et al., 2010, Plant Cell 22: 1620; Zhao and Dixon, 2011, Trends Plant Sci. 16: 227




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Symbolic nomenclature of plant cell wall monosaccharide building blocks

L-Fucose (L-Fuc)

D-Galacturonic acid (D-GalA)

D-Glucuronic acid (D-GlcA)

L-Rhamnose (L-Rha)

D-Aceric acid(D-AceA)

3-deoxy-D-manno-octulosonic acid (Kdo)

3-deoxy-D-lyxo-2-heptulosaric acid (Dha)

D-Xylose (D-Xyl)

L-Arabino furanose (L-Araf)

D-Apiose(D-Api)

Pentoses

Deoxy-hexoses Acidic sugars

L-Arabino pyranose (L-Arap)

Hexoses

LL-Galactose

(L-Gal)

D-Galactose (D-Gal)

D-Glucose (D-Glc)

D-Mannose (D-Man)

Monosaccharide symbols in part from Consortium for Functional Glycomicshttp://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/carbMoleculeHome.jspMohnen, 2008, Curr. Opin. Plant Biol. 11: 266

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Precursors for plant wall polysaccharide formation are synthesized largely via the nucleotide-sugar

interconversion pathway

From: Atmodjo, Hao & Mohnen, 2013, Annu. Rev. Plant Biol. 64: 747

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Cellulose




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The cellulose-hemicellulose network consists of cellulose elementary fibrils and fibers

interacting with matrix polysaccharides

Cellulose

Hemicellulose(Hemicellulose

H-bonds to cellulose and is also present

in the rest of the wall)

Pectin-rich middle lamella


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Cellulose• Most abundant biopolymer in nature

• Makes up ~20-30% of higher plant primary wall and ~50% of secondary wall

• Linear polymer of β-1,4-linked glucose, each residue rotated 180°

Delmer & Amor, 1995, Plant Cell 7: 987-1000; Albersheim et al., 2011, Plant Cell Walls, New York: Garland Sci.

• Hydrogen bonds, hydrophobic interactions between the flat surfaces of the pyranose rings, and Van der Waals interactions hold the chains together to yield crystalline microfibrils

• Length of cellulose microfibrils varies: the degree of polymerization (DP) in primary wall cells falls into two ranges, DP 250-500 and DP 2500-4000; the DP of cellulose in secondary walls is ~10,000-15,000

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• Size of microfibril (2-4 nm diameter, several µm long) varies depending on organism and can range from ~24 or 36 glucan chains in plants up to large fibrils (> 200 chains) in cellulosic algae

• As plant cells mature from primary to secondary walls, cellulose is found associated as macrofibrils or bundles

• The process of macrofibril formation is believed to occurring spontaneously upon microfibril formation

• In cellulose I (type of cellulose found naturally in plants), glucan chains are arranged parallel to each other

Albersheim et al., 2011, Plant Cell Walls, New York: Garland Sci.;Fernandes et al., 2011, PNAS 108: E1195; Guerriero et al., 2010, J. Integrative Plant Biol. 52: 161

The structural units and interactions in cellulose

Single microfibril

Cell wall

Plant cells




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• Genes for plant cellulose synthase catalytic subunits were identified in cotton based on deduced amino acid sequence homology to bacterial cellulose synthase (CesA)

• CESA genes exist in multigene families in plants(e.g. Arabidopsis has 10 CESA genes) and appear to function as rosettes in the plasma membrane

Doblin et al., 2002, Plant Cell Physiol. 43: 1407; Bringmann et al., 2012, Trends Plant Sci. 17: 666

Data support the premise that at least 3 different CESA

proteins are required for a functional CESA complex

Arabidopsis CESAs

Group IPrimary wall

CESAs 1, 3, 6, (2, 5, 9)

Group IISecondary wall CESAs 4, 7, 8

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Freeze fracture replicas of rosettes associated with cellulose microfibril biogenesis

• Rosettes after fracture in leaflet of the plasma membrane bilayer nearest cytoplasm (PF face)

• Several rosettes (surrounded by circles) in plasma membrane of differentiating tracheary element of Zinnia elegans that deposits cellulose into patterned secondary wall thickeningsFrom: Delmer, 1999, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 245

Rosette structures• Containing multiple cellulose

synthase (CESA) proteins have 6-fold symmetry and are associated with cellulose synthesis

• The rosettes move in linear tracks in the plasma membrane and are aligned with cortical microtubules

CESAs reside in rosette structures within the plasma membrane

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A

3 1

11

31

1

1

3 1

11

11

1

1

3

4

4 44

44

4

4

4

CesA1CesA3CesA2, 5, 6, 9

CesA4

CesA7 and CesA8

D E

Rossettes appear to synthesize elementary fibril that contains ~24 to 36 glucan chains

CESA protein and rosette structural models

CesA Rosettesubunit

Rosette

Cellulosemicrofibril

β-1,4-glucanchainC

(C) Doblin et al., 2002, Plant Cell Physiol. 43:1407; From: Atmodjo, 2010, Ph.D. Dissertation, The University of Georgia, as modified from (A) Taylor, 2008, New Phytol. 178: 239; (B) Richmond, 2000, Genome Biol 1: REVIEWS3001; (D) Mutwil, Debolt, & Persson, 2008, Curr. Opin. Plant Biol. 11: 252: (E) Timmers et al., 2009, Plant Physiol. 166: 1465

B




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• Cellulose synthesis occurs at the plasma membrane

• Crystallization of the BcsA-BcsB cellulose synthase complex from the bacterium Rhodobacter sphaeroides suggests that the growing cellulose chain is elongated and translocated one glucose unit at a time with the direction of rotation changing 180º between each glucose unit addition

Proposed model for cellulose synthesis and translocation in bacteriaFrom: Morgan et al., 2013, Nature 493: 181

For model of plant cellulose synthase see Latsavongsakda et al., 2013, PNAS 110: 7512

Bacterial cellulose synthase complex has been crystallized, providing hints

for function of plant CESAs and rosettes

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• Hypothesized that assembly of CESA complexes takes place in Golgi and that the complexes may be transported to the plasma membrane in Golgi-derived vesicles known as small CESA compartments (smaCCs) or microtubule-associated cellulose synthase compartments (MASCs)

• Proteins that influence cellulose synthesis but whose specific function in cellulose synthesis remains unclear include:

– UDP-Glc synthesizing proteins (SUSY)

– Membrane-bound endo-beta-1,4-glucanase KORRIGAN

– Proteins involved in cellulose synthesis and expansion (COBRA, COBRA-LIKE; KOBITO/ELD1)

– Cytoskeleton-related proteins (Microtubule-related DRP1A, POM2/CSI1, POM1/CTL1, annexins, actin, tubulin)

• In planta cellulose microfibrils complex with hemicellulosic polysaccharides such as xyloglucan

Bringmann et al., 2012, Trends Plant Sci. 17: 666; Crowell et al., 2009, Plant Cell 21: 1141; Gutierrez et al., 2009, Nat. Cell Biol. 11: 797

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Scanning electron micrograph of an untreated cellulose strand

mechanically extracted from corn husk

Scanning electron micrograph of a partially purified fiber bundle

from corn husks

“In their natural state, and before chemical extraction, fiber surfaces have waxes and other encrusting substances

such as hemicellulose, lignin and pectin that form a thick outer layer to protect the cellulose inside”

From: Reddy & Yang, 2005, Trends Biotechnol. 23: 22




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Hemicellulose

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Hemicelluloses

• Have backbone of β-(1,4)-linked sugar residues with an equatorial configuration at C1 and C4

Hemicellulosic polysaccharides:

• Xyloglucan

• Mixed-linkage glucan

• Mannan and glucomannan

• Xylan

From: Scheller & Ulvskov, 2010, Annu. Rev. Plant Biol. 61: 263

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4)- β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,4)-β-D-Glcp-(1,

Hemicelluloses - xyloglucan

• A major hemicellulose (up to 25%) in type I primary walls; minor component (<2%) in type II primary walls

1↓2

α-L-Fuc

1↓6

α-D-Xyl1↓6

α-D-Xyl1↓6

α-D-Xyl1↓6

α-D-Xyl1↓6

α-D-Xyl1↓6

α-D-Xyl

X X X G GX L F

β4 β4 β4 β4 β4 β4 β4α6 α6 α6

β2

α2

α6 α6 α6

X X X G GX L F

β2

• Most plants synthesize XXXG-type XyG (includes XXXG, XXFG, XXLG, XLFG)

• Commelinid monocots synthesize predominantly non-fucosylated XXGn-type XyG (e.g. XXGG, XXGGG)

1↓2

β-D-Gal1↓2

β-D-Gal May be mono- or di-acetylated

Reviewed in Zabotina, 2012, Front. Plant Sci. 3: 134




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• Three different views of the predicted cellulose-like flat glucan backbone conformation of a XG17 fragment of xyloglucan

• The flat backbone conformation can be adopted on binding of xyloglucan to cellulose microfibrils

Adoption of xyloglucan into a flat glucan backbone conformation would allow H-bonding

of xyloglucan to cellulose microfibrils

From: Levy et al., 1991, Plant J. 1: 195

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However, there is structural diversity in xyloglucan sidechain structure

Xyloglucan sidechains have variable structure depending on the source of the walls from which the xyloglucan is isolated; (a) The species from which the xyloglucan was isolated;

(b) A single letter abbreviation used to designate specific XG structures (Fry et al., 1993, Physiol. Plantarum 89: 1)

Fry et al., 1993, Physiol. Plantarum 89: 1; York et al., 1996, Carbohydr. Res. 285: 99; Hantus et al., 1997, Carbohydr. Res. 304: 11; Ray et al., 2004, Carbohydr. Res. 339: 201; Pena et al., 2008, Glycobiology 18:891; Pena et al., 2012, Plant Cell 24: 4511

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Hemicelluloses - mixed linkage glucan & mannan

β4 β4 β4 β4 β4 β4 β4α6

4)-β-D-Manp-(1,4)-β-D-Glcp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Glcp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,

1↓6

α -D-Galp

(Galacto)glucomannan• Major component of secondary wall in gymnosperms• Constituents of bulbs, tubers, seeds, roots, and leaves

in some monocot plants e.g. Aloe vera, voodoo lily

β4 β4 β4 β4 β4 β4 β4α6 α6 α6 α6

4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,4)-β-D-Manp-(1,

1↓6

α -D-Galp1↓6

α -D-Galp1↓6

α -D-Galp1↓6

α -D-Galp

Galactomannan• Abundant in cell walls of storage tissues of seeds

notably in legumes

Ebringerová, Hromádková, & Heinze, 2005, Adv. Polym. Sci 186: 1; Scheller & Ulvskov, 2010, Annu. Rev. Plant Biol. 61: 263

[β -D-Glcp-(1,4]n-β -D-Glcp-(1,3)-β -D-Glcp-(1,4)]m, where n and m are 3 or 4

Mixed linkage glucan (β-1,3/β-1,4 glucan)• ~70% (1,4)-linked and 30% (1,3)-linked D-Glc• Present in grasses (Poaceae)

and Equisetum (horsetail)




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Hemicelluloses - xylan

4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,

1↓2

4-O-Me-α-D-GlcpA1↓2

α -D-GlcpA

↓O-acetyl

Glucuronoxylan (GX) in dicots• May also have Araf substitution to a lesser extent

β4 β4 β4 β4 β4 β4 β4

α2 α2Ac

β4

4M

4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,3)-α-L-Rhap-(1,2)-α-D-GalpA-(1,4)-β-D-Xylp

β4 β3 α2 α4

Xylan reducing end sequence identified in dicots and Gymnosperms

4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,

1↓3

α-L-Araf1

3

α-L-Araf1

2

α-L-Araf

1↓5

Feruloyl

5Fer

β4 β4 β4

α3α3

α2

Arabinoxylan (AX)in cereal grains


β4 β4 β4

α3α3

α2

5Fer

α2

4M β2

1↓3

α -L-Araf1↓3

α -L-Araf

α -L-Araf

1↓2

β-D-Xylp

4-O-Me-α-D-GlcpA

2↑1

2↑1

Glucuronoarabinoxylan (GAX) in grasses

Glucuronoarabinoxylan (GAX) in Gymnosperms

β4 β4 β4

α3 α2

4M

α -L-Araf 4-O-Me-α-D-GlcpA1↓3

1↓2


Ebringerová, Hromádková, & Heinze, 2005, Adv. Polym. Sci 186: 1; York & O’Neill, 2008, Curr. Opin. Plant Biol. 11: 258

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Cellulose synthase-like (Csl) genes mediate synthesis of the backbones of hemicelluloses

Modified from Davis et al.,2010, Plant J. 64: 1028

AtCSLC4AtCSLA9

Farrokhi et al., Plant Biotechnology Journal 4(2): 145-167

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Xyloglucan biosynthesis

β4 β4 β4

β2

α2

α6 α6 α6

GX L F

β2

Modified from Oikawa et al., 2013, Trends Plant Sci. 18: 49

O-acetyl

* Catalytic activity has been demonstrated

CSLC4*

XXT1*XXT2*XXT3XXT4*XXT5

MUR3*†

XLT2† FUT1/MUR2*AXY4, AXY4L

Reviewed in: Zabotina, 2012, Front. Plant Sci. 3: 134; Pauly et al., 2013, Planta 238: 627

† XUT1, a homolog of MUR3 and XLT2, incorporates GalA residues (instead of Gal) into XyG in Arabidopsis root hair




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Mixed-linkage glucan biosynthesis

• Expression of the rice and barley CSLF and CSLH genes in Arabidopsis and tobacco result in synthesis of mixed-linkage glucan in these dicotyledonous plantsBurton et al., 2006, Science 311: 1940; Doblin et al., 2009, PNAS 106: 5996; Vega-Sánchez et al., 2012, Plant Phys, 159: 56

OsCSLF2*, 4*, & 6*HvCSLH1*


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Mannan and glucomannan biosynthesis

β4 β4 β4 β4 β4 β4 β4α6 α6 α6 α6

• Membrane-bound α-1,6-galactosyltransferase*Identified from seed endosperm of fenugreek (Trigonella foenum-graecum L)

β4 β4 β4 β4 β4 β4 β4α6

Reviewed in: Scheller & Ulvskov, 2010, Annu. Rev. Plant Biol. 61: 263; Verhertbruggen et al., 2011, Plant Signal. Behav. 6: 10; Liepman & Cavalier, 2012, Front Plant Sci. 3: 109; Pauly et al., 2013, Planta 238: 627

• Multiple CSLA family members* from Arabidopsis, poplar, guar, voodoo lily, rice, loblolly pine, and mosse.g. AtCLSA1, AtCSLA2, AtCSLA9 have both mannan and glucomannan synthase activities, while AtCSLA7 was shown to have only mannan synthase activity


AtCSLD2, 3, and 5??

• AtMSR1, AtMSR2, and TfMSR (mannan-synthesis related)have also been implicated in mannan synthesis, however their exact roles are still unclear

51

Xylan biosynthesis

β4 β4 β4

α3α3

α2

5Fer

α2

4M β2 OsXAX1

TaXAT1, TaXAT2OsXAT2, OsXAT3

Os PF02458 family?

Glucurono-arabinoxylan

in grasses


β4 β4 β4 β4 β4 β4 β4

α2 α2

2Ac

β4

4M

β3 α2 α4

AtGUX1*AtGUX2-4

AtGXMT1/GXM3* AtGXM1*, AtGXM2*AtIRX15? ATIRX15-L?

β4

Reducing end sequence

??

AtIRX8/GAUT12?AtIRX7/FRA8?AtIRX7-L/F8H?

AtPARVUS/GATL1?

Xylan backboneAtIRX9, AtIRX10, AtIRX14AtIRX9-L, AtIRX10-L, AtIRX14-L• Note: Co-expression of AtIRX9 + AtIRX14 in tobacco

has been reported to yield xylosyltransferase activity

3Ac

AtESK1/TBL29

OsIRX9, OsIRX9-L, OsIRX14

Reviewed in: Scheller & Ulvskov, 2010, Annu. Rev. Plant Biol. 61: 263; Pauly et al., 2013, Planta 238: 627

Glucuronoxylanin dicots




52

Pectin

53

The pectic polysaccharides exist in the middle lamella and throughout the wall

Middlelamellae

Pectins are present in the middle lamella

and in the rest of the primary wall


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Pectin

A family of polysaccharidesthat contain α-D-galacturonic acid (GalA)

linked at both the 1 and 4 positions

Pectic polysaccharides:

• Homogalacturonan (HG)

• Xylogalacturonan (XGA)

• Apiogalacturonan (AGA)

• Rhamnogalacturonan I (RG-I)

• Rhamnogalacturonan II (RG-II)




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Pectin - homogalacturonan (HG),xylogalacturonan (XGA), and apiogalacturonan (AGA)

[4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,]n

Homogalacturonan• Partially methylesterified at O-6• Partially O-acetylated at O-2 and/or O-3

α4 α4 α4 α4-4 α4 α4 α4 α4 α4 α4 α4 α-6M 6M 6M 6M 6M

2Ac3Ac2Ac

Xylogalacturonan• Side chain Xyl may be further elongated at O-2 by another Xyl

α4 α4 α4 α4 α4 α4 α-β3 β3β3 β3

α4-4

4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,

1↓3

β-D-Xylp1↓3

β-D-Xylp1↓3

β-D-Xylp1↓3

β-D-Xylp

Apiogalacturonan• Found in aquatic plants e.g. duckweed (Lemna minor)

α4 α4 α-α4 α4 α4-42 or 3

β3

2 or 3

4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,

1↓

2 or 3

D-Apif1↓

2 or 3

D-Apif

1↓3

β-D-Apif

Reviewed in: Caffall & Mohnen, 2009, Carbohydr. Res. 344: 1879; Atmodjo, Hao & Mohnen, 2013, Annu. Rev. Plant Biol. 64: 747

56

Pectin - rhamnogalacturonan II (RG-II)

4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,4)-α -D-GalpA-(1,

1↓5

2↓3

α -L-Rhap

α -D-Kdo

1↓5

2↓3

β -L-Araf

β -D-Dha

Side chain A

Side chain D Side chain C

Side chain B

2↑1

β -D-Apif

α -D-GalpA 1→2β -L-Rhap3←1β-D-GalpA

3’↑1

4↑1

2-O-Me α -D-Xylp1→3α -L-Fucp

2↑1

4↑1

β -D-GlcpA

L-Galp

2↑1

β -D-Apif

β -L-Rhap

3’↑1

3↑1

±AcO→ α -L-AcefA

2↑1

2↑1

β -D-Galp 4←1α -L-Arap

2-O-Me α -L-Fucp ←OAc ±

1-α -L-Rhap

1-α -L-Rhap 2←1β -L-Araf

32

L

2M

2M

57

2 αβ4

α3

α2

α5

2 αβ4

α3α4

2 αβ4

β4

β6

2 αβ4

β6

2 αβ4

β4

α2

2 α4

5

2 α4

36

2 α4

26

(Deduced)

α5

α5

α5

α5

α5

α2

α3

α3

α2

2 αα4

[4)-α -D-GalpA-(1,2)-α-L-Rhap-(1,4)-α-D-GalpA-(1,2)-α-L-Rhap-(1,]n

α2 α4 α2 α4 α2 α4 α2 α4 α2 α4 α2 α--42Ac 3Ac 3Ac3Ac2Ac

Pectin - rhamnogalacturonan I (RG-I)Backbone

• Most or all of GalA residues are O-acetylated at O-2 or O-3; Acetylation of Rha has also been reported

• 25-80% Rha is substituted at O-4 by side chains

Side chains

• Size of individual side chains can vary from one to >30 sugar residues

• Predominantly contains arabinosyl and galactosyl residues

• Galactan-containing side chains may be terminated by single α-L-Fuc, β-D-GlcA, or 4-O-Me-β-D-GlcA residues

Albersheim et al., 2011, Plant Cell Walls, New York: Garland Sci.; McNeil, Darvill, & Albersheim, 1982, Plant Physiol. 70: 1586; Zheng & Mort, 2008, Carbohydr. Res. 343: 1041




58

Interconnection between different pectic domains

Within pectic polysaccharides

In pectin-containing proteoglycan APAP1


AGP AG

59

Homogalacturonan biosynthesis

• Arabidopsis GAUT1:GAUT7 HG:α-1,4-galacturonosyltransferase (GalAT) complex synthesizes the HG backbone


α4 α4 α4 α4-4 α4 α4 α4 α4 α4 α4 α4 α-

6M 6M 6M 6M 6M

2Ac3Ac2Ac

GAUT1*:GAUT7 complexCGR3, QUA2, QUA3


• Mutants of Arabidopsis TBR, TBL3, and PMR5 have reduced level of pectin esterification as measured by Fourier transform infrared (FTIR) microspectroscopyVogel et al., 2004, Plant J. 40: 968; Bischoff et al., 2010, Plant Physiol. 153: 590

60

Xylogalacturonanand rhamnogalacturonan II biosynthesis

α4 α4 α4 α4-4 α4 α4 α4 α4 α4 α4 α4 α-β3 β3 β3

β2 XGD1*Xylogalacturonan

Rhamnogalacturonan II

RGXT1-4*

Reviewed in: Atmodjo, Hao & Mohnen, 2013, Annu. Rev. Plant Biol. 64: 747

* Catalytic activity has been demonstratedL

2M

2M




61

Rhamnogalacturonan I (RG-I) biosynthesis

• ARAD1 and ARAD2 were shown by BiFC, FRET, and non-reducing SDS-PAGE to form homo- and heterodimers mediated by disulfide bonds, suggesting possible protein complex involvement in RG-I arabinan synthesis

α2 α4 α2 α4 α2 α4 α2 α4 α2 α4 α2 α--4

Reviewed in: Atmodjo, Hao & Mohnen, 2013, Annu. Rev. Plant Biol. 64: 747

α5

α5

α5

α5

α5

α2

α3

α3

α4

α2

ARAD1ARAD2

*Catalytic activity has been demonstrated

β4

β4

β4

β4GALS1*GALS2GALS3

62

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 wall

Research is needed to understandcell wall architecture and its synthesis

63From: Tan et al., 2013, Plant Cell 25: 270

For example, the recently discovered proteoglycan APAP1 provides one example of how generally considered separate wall matrix polysaccharides

are connected in one covalently linked structure




64

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

65

Additional references

• Mortimer et al., 2010, PNAS 107: 17409-14• Rennie et al., 2012, Plant Physiol. 159: 1408-17

• Scheller & Ulvskov, 2010, Annu. Rev. Plant Biol. 61: 263-89

• Urbanowicz et al., 2012, PNAS 109: 14253-8

• Wu et al., 2009, Plant J. 57: 718

• Wu et al., 2010, Plant Physiol. 153: 542• Xiong et al., 2013, Mol. Plant 6: 1373

• Yuan et al., 2013, Plant Cell Physiol. 54: 1186

• CAZy database– Cantarel et al., 2009, Nucleic Acids Res. 37: D233

• Hemicellulose synthesis– Galactomannan synthesis

• Reid et al., 2003, Plant Physiol. 131: 1487

– Xylan synthesis• Anders et al., 2012, PNAS 109: 989-93

• Bromley et al., 2013, Plant J. 74: 423• Brown et al., 2011, Plant J. 66: 401-13

• Chiniquy et al., 2012, PNAS 109: 17117-22

• Chiniquy et al., 2013, Front Plant Sci. 4: 83

• Faik, 2010, Plant Physiol. 153: 396-402

• Jensen et al., 2011, Plant J. 66: 387-400• Lee et al., 2012, Plant Cell Physiol. 53: 1204-16

• Lee et al., 2012, Plant Cell Physiol. 53: 1934-49

• Lee, Zhong, & Ye, 2012, Plant Cell Physiol. 53: 135-43

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Acknowledgements

PhotographyStefan Eberhard - CCRC

Funding AgenciesU.S. Department of Agriculture - AFRI/NIFA

National Science FoundationU.S. Department of Energy - BioEnergy Science Center (BESC)

Complex Carbohydrate Research Center

The University of Georgia, Athens




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Plant Cell Wall Structure and Biosynthesis Prof. Debra