Matrix polysaccharide biosynthesis: Xyloglucans and Pectins

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Matrix polysaccharide biosynthesis:

Xyloglucans and Pectins

Matrix polysaccharide biosynthesis:

Xyloglucans and Pectins

Michael G. Hahn University of Georgia BioEnergy Science Center

Complex Carbohdyrate Research Center Athens, GA, USA

Michael G. Hahn University of Georgia BioEnergy Science Center

Complex Carbohdyrate Research Center Athens, GA, USA

Advanced School on Biochemistry of Biofuels Itamambuca, Brazil

September 28, 2010

Advanced School on Biochemistry of Biofuels Itamambuca, Brazil

September 28, 2010

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Model of the cellulose/hemicellulose and pectic cell wall networks in primary walls

[McCann & Roberts (1991) The Cytoskeletal Basis of Plant Growth and Form, p. 126]

(3)

Sugar nucleotide

synthesis/metabolism as a

target for manipulation

(4)

ADP-α-D-Glc

Ara-5-P GDP-β-L-Fuc GDP-4k-6d-D-Man

GDP-α-D-Man GDP-L-Gul

GDP-^L-Gal

UDP-β-L-Rha

UDP-α-^D-Glc UDP-α-^D-Gal

UDP-4k-6d-^D-Glc β-L-Rha-1-P

L-Rha

UDP-α-D-GlcA UDP-α-D-GalA

UDP-α-D-Xyl UDP-D-Api

UDP-L-Arap

α-D-Glc-1-P

D-Frc-6-P

D-Glc-6-P ^D-Glc Frc D-Man-6-P ^D-Man α-D-Man-1-P

GlcN-6-P GlcNac-1-P

UDP-α-D-GlcNac

ADP-^D-Gal β-L-Fuc-1-P

L-Fuc

ATP ADP GTP PPi

PPi UTP CoA AcCoA

Gln Glu PPi GTP

Gal-1-P Glc-1-P

ATP PPi

UTP PPi ADP

Frc

UDP Frc TDP

TDP-D-Glc Frc

ATP ADP UTP PPi

TDP-β−L-Rha

α-D-Gal-1-P ADP

ATP UTP PPi

D-Gal

O₂

α-D-GlcA-1-P

ATP ADP UTP PPi

L-Ara-1-P

ATP ADP UTP PPi

L-Ara

α-D-Xyl-1-P UTP PPi Xyl

D-GlcA

α-D-GalA-1-P ADP

ATP UTP PPi

D-GalA

KDO-8-P

KDO CTP

CMP-KDO PPi UDP-α-D-sulfoQuin

R-SO₃ R

PEP

CO₂

CO₂ 2

3

3 3

4

6 5 7 8 10 9

10

11 12 13

13

13 14

15

16 17

18 13

19 20

13 1 21 23 22

24 25

27 26

29 30

ADP ATP 31 ADP ATP

31

34

35

36 TDP-D-GlcA

4

GDP-D-Glc GTP37 PPi

GTP PPi

UDP-β-L-Araf L-Gal-1-P

L-Gal

38 40 39

GDP Pi 40 41 43

42

Inositol Inositol Ascorbate

ADP ATP 32 ADP ATP

32 ATP ADP

33 ATP ADP

33

NAD⁺ NADH Sucrose

Sucrose

44

Glc Glc6P

Glc6P

Frc

Glc Sucrose-6-P

46 45 Frc6P UDP-Glc

mbp

The metabolism of NDP-sugars in plants: plant cell wall precursors.

Mohnen, Bar-Peled and Somerville (2008).

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Biosynthetic pathway for GDP-L-fucose in Arabidopsis thaliana

GMD1

GMD2 (mur1)

GER1 GER2?

[Adapted from: Bonin et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2085-2095]

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Mutation in GMD2 (mur1) results in reduced fucosylation in the non-meristematic regions of the root

CCRC-M1 GMD1::GUS

Bonin et al. (2003) Freshour et al. (2003)

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Xyloglucan biosynthesis

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Reverse genetic approaches have identified several genes encoding glycosyltransferases involved in

xyloglucan biosynthesis

Reverse genetic approaches have identified several genes encoding glycosyltransferases involved in

xyloglucan biosynthesis

FUT1 (MUR2)

MUR3

XXT1 XXT2 XXT5 CSLC

[Lerouxelet al., Curr. Opin. Plant Biol. (2006) 9:621-630]

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Oligosaccharide content of wild-type and mur2 xyloglucans Oligosaccharide content of wild-type and mur2 xyloglucans

[Vanzin et al. (2002) Proc. Natl. Acad. Sci. 99:3340-3345]

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Phylogenetic tree of putative xylosyl- and glycosyl- transferases belonging to CaZY Family GT34

[Zabotina et al. (2008) Plant J. 56:101-115]

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Localization of xyloglucan epitopes in wild-type and xylosyltransferase mutants of Arabidopsis

[Cavalier et al. (2008) Plant Cell 20:1519-1537]

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Oligosaccharide content of wild-type and mur3 xyloglucans Oligosaccharide content of wild-type and mur3 xyloglucans

[Madson et al. (2003) Plant Cell 15:1662-1670]

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Pectin biosynthesis

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Schematic structure of pectin showing four pectic polysaccharides rhamnogalacturonan I (RG-I), xylogalacturonan (XGA),

homogalacturonan (HG), and rhamnogalacturonan II (RG-II) linked to each other.

RG-I XGA HG

RG-II

[Mohnen(2008) Curr. Opin. Plant Biology]

HG should be increased ~12.5-fold and RG-I increased ~2.5-fold to approximate amounts of these polysaccharides in walls. Monosaccharide symbols taken in part from Symbol and Text Nomenclature for

Representation of Glycan Structure from the Consortium for Functional Glycomics

(http://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/carbMoleculeHome.jsp).

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Pectic polysaccharide

backbone biosynthesis

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[Yin et al., Plant Physiol. (2010) 153:1729-1746]

Phylogenetic tree of CaZY Family GT8 indicates two distinct families of plant GT8 proteins

Three major classes of GT8 proteins in nature

A putative progenitor of cell wall-related GT8 genes in plants Cell wall-related GT8

proteins in plants

Non-cell wall GT8 proteins in plants

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Arabidopsis contains a 25 member GAUT1-related gene family

14 genes with

52-81% similarity and 36-68% identity to GAUT1.

These 15 genes named the

GAlactUronosylTransferase (GAUT)-related family.

Additional 10 genes with

42-52% similarity and 23-29% identity to GAUT1.

Named the GAUT-Like (GATL) family.

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estExt_Gw1Plus.C_1200026

eugene3.01370031 estExt Gw1Plus.C LG XIV0504AtGAUT1

Os09g36180

AtGAUT4

fg4_pg.C_LG_VI000014 Os08g23780

Os08g38740

Os09g30280 e gw1.XVI.562.1

estExt Gw1Plus.C 281089

Os05g40720

Os12g02910 Os11g03160

Os03g21250 Os07g48370 Os11g37980

eugene3.00051260

fg4 pg.C LG II000411 AtGAUT5 AtGAUT12

estExt fg4 pm.C LG XIII0357 Os03g30000

AtGAUT11 eugene3.01290051

grail3.0138001201 Os04g54360AtGAUT10 fg4 pm.C LG XIII000435

eugene3.00080075 eugene3.00002521

AtGAUT8 Os02g29530

eugene3.03090007 AtGAUT9

Os06g12280 Os02g51130

Os10g21890 Os06g51160

AtGAUT2 Os06g49810 Os09g36190

eugene3.00021408

B-1

A-1

A-2

A-3

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0.5 eugene3.00111083

eugene3.00041059

AtGAUT14 Os12g38930 Os03g11330 Os01g52710

estExt_Gw1Plus.C_LG_XIV2539 AtGAUT15

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(79) A-4

B-2

C

AtGAUT6

AtGAUT7

Comparative phylogenetic analysis of Arabidopsis thaliana, Populus trichocarpa and Oryza sativa GAUTs

[Caffall et al. (2009) Molecular Plant 2:1000-1014]

AtGAUT1 HG:GalAT

AtGAUT7 AtGAUT8

Qua1

?HG:GalAT

AtGAUT12 Irx8

?HG:GalAT or Xylan Primer/Cap?

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GAUT1 & GAUT7

Type II transmembrane protein (Keegstra and Raikhel. 2001.

Curr. Opin. Plant Biol. 4:219-224)

• Predicted Type II transmembrane proteins

• Glycosyltransferase Family 8

(CAZy database - http://afmb.cnrs-mrs.fr/CAZY/)

• Predicted GT-A structure

• Golgi residents (Dunkley et al., 2004, 2006)

673 a.a.

77.4 kDa pI 9.95 GAUT1

(At3g61130)

619 a.a.

69.7 kDa pI 8.63 GAUT7

(At2g38650)

transmembrane domain

globular / catalytic domain

N-terminal tail stem

[from Debra Mohnen] (Topology prediction using HMMTOP v.2)

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ExoPG Expt.

Initial SP protein GAUT1 depleted GAUT1 enriched

G

0 2 4 6 8 10 12 14 16

Water Boiled EPG EPG

pmolGalAincorp.

Sensitivity of product produced by GAUT1- immunosorbed enzyme to expolygalacturonase

proves synthesis of HG

Sterling et al., (2006) PNAS

GAUT1 is a HG-GalAT

Not known if GAUT1 is involved in initiation and/or elongation phase of HG synthesis.

[GalAT activity in SP-Fraction can NOT de novo synthesize HG i.e. UDP-GalA + GAUT1 can not de novo make oligogalacturonides]

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Heterologously expressed GAUT7 does not have HG:GalAT activity

GAUT7

GAUT1

[Sterling et al. (2006) Proc. Natl. Acad. Sci. 103:5236-5241]

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Mutation in GAUT8 (QUA1) affects morphology and cell adhesion

[Bouton et al. Plant Cell (2002) 14: 2577-2590]

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Mutation in GAUT8 (QUA1) affects both GalA and Xyl content of Arabidopsis stem cell walls

[Orfila et al. Planta (2005) 222: 613-622]

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[Kong et al., Plant Physiol. (2010), submitted ]

GATL- a

GATL-f

GATL-e

AG LT

-d

GA TL-c GATL-b

AG LT

-g

Phylogenetic tree of the GATL family

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Pairs of GATL genes that are associated with genome duplication events in Arabidopsis

Recent dupl.

Older dupl.

[Kong et al. Plant Physiol. (2010) submitted]

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Plant growth and xylem/fiber morphology are altered in the gatl1/parvus/glz mutant

if

if Ve

Ve Ph

Ph

Ve

Ve A

B

C

D

Fiber

Xylem vessel

Xylary fiber

Vessel

Stem

Hypcotyl

w.t. gatl1

[Kong et al., unpublished]

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Expression of PdGATL1.1 and PdGATL1.2 genes in gatl1/parvus/glz plants restores w.t. growth phenotypes

WT gatl1 1 2 3 4 5 6 gatl1 mutants transformed

with PdGATL1.1

gatl1 mutant with PdGATL1.1

gatl1 mutant with PdGATL1.2

T-DNA insertion gatl1 mutant

Wild Type

Endogenous AtGATL1 gene PdGATL1.1 transgene PdGATL1.1 transcript

ACTIN

gatl1 mutants transformed with PdGATL1.2 WT gatl1 1 2 3 4 5 6 T-DNA

insertion Endogenous AtGATL1 gene PdGATL1.2 transgene PdGATL1.2 transcript ACTIN

[Kong et al. (2009) Mol. Plant 2:1040-1050]

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Expression of PdGATL1 genes in gatl1/parvus/glz plants mostly restores w.t. tissue morphology

[Kong et al. (2009) Mol. Plant 2:1040-1050]

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Mutation of GATL5 leads to reduced mucilage synthesis in developing seed of Arabidopsis

WT gatl5-1 proGATL5::GATL5 (gatl5-1)

[Kong et al. (2010), in preparation]

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0 5 10 15 20 25 30 35 40 45

GATL5 GATL6

relative fold change

4 DPA 7 DPA 10 DPA

GATL5 and GATL6 transcript analysis in developing seed of Arabidopsis

[Kong et al. (2010), in preparation]

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Pectic polysaccharide

side-chain biosynthesis

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Mutation in ARAD1 results in reduced Ara content in

Arabidopsis leaf and stem and alters rhamnogalacturonan I structure

Mutation in ARAD1 results in reduced Ara content in

Arabidopsis leaf and stem and alters rhamnogalacturonan I structure

[Harholt et al. Plant Physiol. (2006) 140: 49-58]

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Mutation in XGD1 results in reduced Xyl content in Arabidopsis pectin but not in xylan/xyloglucan Mutation in XGD1 results in reduced Xyl content in

Arabidopsis pectin but not in xylan/xyloglucan

Cell wall composition

[Jensen et al. Plant Cell (2008) 20:1289-1302]

Pectin

composition

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What about ALL of the other types of enzymes that we know

MUST

be involved in synthesis of pectic

polysaccharides

What about ALL of the other types of enzymes that we know

^MUST

be involved in synthesis of pectic

polysaccharides

What about other glycosyl transferases necessary to complete the synthesis of Rhamnogalacturonan I?

Fucosyltransferases (FUT4, FUT6) that adds terminal fucosyl residues to arabinogalactan acceptors have been identified (Wu et al., 2010).

What about Rhamnogalacturon-II side-chain biosynthesis?

Several genes have been identified that encode proteins that can transfer xylose to fucosyl residues (Englund et al., 2006; 2008).

What about the many other unusual glycosyl residues in RG-II?

What about non-carbohydrate modifications (e.g., methyl, acetyl groups)?

Putative pectin methyltransferase (QUA2/TSD2) identified that have a

“methyltransferase domain” appears to be involved in methyl esterification of homogalacturonan (Mouille et al., 2007; Krupkova et al., 2007).

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Interdependence of the cellulose/hemicellulose and pectic cell wall networks in primary walls Interdependence of the cellulose/hemicellulose

and pectic cell wall networks in primary walls

[McCann & Roberts (1991) The Cytoskeletal Basis of Plant Growth and Form, p. 126]