Matrix polysaccharide biosynthesis: Xyloglucans and Pectins

Matrix polysaccharide biosynthesis: Xyloglucans and Pectins

Michael G. HahnUniversity of Georgia

BioEnergy Science CenterComplex Carbohdyrate Research Center

Athens, GA, USA

Michael G. HahnUniversity of Georgia

BioEnergy Science CenterComplex Carbohdyrate Research Center

Athens, GA, USA

Advanced School on Biochemistry of BiofuelsItamambuca, BrazilSeptember 28, 2010

Advanced School on Biochemistry of BiofuelsItamambuca, BrazilSeptember 28, 2010

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

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

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

Sugar nucleotide synthesis/metabolism as a

target for manipulation

ADP-α-D-Glc

Ara-5-P

GDP-β-L-Fuc GDP-4k-6d-D-ManGDP-α-D-Man

GDP-L-Gul

GDP-L-Gal

UDP-β-L-Rha

UDP-α-D-GlcUDP-α-D-Gal

UDP-4k-6d-D-Glcβ-L-Rha-1-PL-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-PGlcNac-1-PUDP-α-D-GlcNac

ADP-D-Gal

β-L-Fuc-1-PL-FucATP ADP GTP PPi

UTPPPi AcCoACoA

GlnGlu

GTPPPi

Gal-1-PGlc-1-P

ATP

PPi

UTPPPi

ADPFrc

UDP

Frc

TDPFrcTDP-D-Glc

ADPATP PPiUTP

TDP-β−L-Rha

α-D-Gal-1-PADPATP PPiUTP

D-Gal

O2

α-D-GlcA-1-PADPATP PPiUTP

L-Ara-1-PADPATP PPiUTP

L-Ara

α-D-Xyl-1-PPPiUTP

Xyl

D-GlcA

α-D-GalA-1-PADPATP PPiUTP

D-GalAKDO-8-P

KDOCTP

CMP-KDO

PPi

UDP-α-D-sulfoQuin

R-SO3R

PEP

CO2

CO2

23

3

3

4

56

7

8

910

10

11 12 13

13

13

13 14

15

16

17

18 13

19 2013

1

21

2223

2425

262729 30

ATPADP

31

ATPADP

31

34

35

36

TDP-D-GlcA

4

GDP-D-Glc37GTP PPiGTP PPi

UDP-β-L-Araf

L-Gal-1-PL-Gal

38

3940

GDPPi40

41

43

42

InositolInositol

Ascorbate

ATPADP

32

ATPADP

32

ATPADP

33

ATPADP

33

NAD+

NADH

SucroseSucrose

44

GlcGlc6P

Glc6P

Frc

GlcSucrose-6-P

4546

Frc6PUDP-Glc

mbp

The metabolism of NDP-sugars in plants: plant cell wall precursors.Mohnen, Bar-Peled and Somerville (2008).

Biosynthetic pathway for GDP-L-fucose in Arabidopsis thaliana

GMD1GMD2 (mur1)

GER1GER2?

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

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

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

CCRC-M1 GMD1::GUS

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

Xyloglucan biosynthesis

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

XXT1XXT2XXT5

CSLC

[Lerouxelet al., Curr. O

pin. Plant B

iol. (2006) 9:621-630]

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

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

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

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

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

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

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

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

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

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

Pectin biosynthesis

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 HGRG-II

[Mohnen

(2008) Curr. O

pin. Plant B

iology]

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).

Pectic polysaccharide

backbone biosynthesis

[Yin

et a

l., P

lant

Phy

siol

. (20

10) 1

53:1

729-

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

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.

estExt_Gw1Plus.C_1200026

eugene3.01370031

estExt Gw1Plus.C LG XIV0504AtGAUT1Os09g36180

AtGAUT4fg4_pg.C_LG_VI000014Os08g23780

Os08g38740

Os09g30280 e gw1.XVI.562.1estExt Gw1Plus.C 281089

Os05g40720

Os12g02910Os11g03160

Os03g21250

Os07g48370

Os11g37980

eugene3.00051260

fg4 pg.C LG II000411

AtGAUT5

AtGAUT12estExt fg4 pm.C LG XIII0357

Os03g30000AtGAUT11

eugene3.01290051grail3.0138001201

Os04g54360AtGAUT10

fg4 pm.C LG XIII000435

eugene3.00080075eugene3.00002521

AtGAUT8

Os02g29530

eugene3.03090007AtGAUT9

Os06g12280Os02g51130

eugene3.00660198AtGAUT3

Os10g21890Os06g51160 AtGAUT2

Os06g49810

Os09g36190

eugene3.00021408

B-1

A-1

A-2

A-3

(60)

0.5eugene3.00111083

eugene3.00041059eugene3.00170460

AtGAUT13AtGAUT14

Os12g38930Os03g11330Os01g52710

estExt_Gw1Plus.C_LG_XIV2539AtGAUT15

(67)

(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]

AtGAUT1HG:GalAT

AtGAUT7

AtGAUT8Qua1?HG:GalAT

AtGAUT12Irx8?HG:GalAT or XylanPrimer/Cap?

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 kDapI 9.95

GAUT1 (At3g61130)

619 a.a.69.7 kDapI 8.63

GAUT7 (At2g38650)

transmembranedomain

globular / catalyticdomain

N-terminal tail stem

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

ExoPG Expt.

Initial SP protein GAUT1 depleted GAUT1 enriched

G

0

2

4

6

8

10

12

14

16

WaterBoiled EPGEPG

pmol

Gal

Ain

corp

.Sensitivity of product produced by GAUT1-

immunosorbed enzyme to expolygalacturonaseproves synthesis of HG

Sterling et al., (2006) PNAS

GAUT1 is a HG-GalATNot known if GAUT1 is involved in initiation and/or elongation phase of HG synthesis.

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

Heterologously expressed GAUT7 does not have HG:GalAT activity

GAUT7

GAUT1

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

Mutation in GAUT8 (QUA1) affects morphology and cell adhesion

Mutation in GAUT8 (QUA1) affects morphology and cell adhesion

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

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

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

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

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

GATL-a

GATL-f GATL-e

GA

TL-dGATL-c

GATL-b

GA

TL-g

Phylogenetic tree of the GATL family

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

Recent dupl.

Older dupl.

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

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

Xylaryfiber

Vessel

Stem

Hypcotyl

w.t. gatl1

[Kong et al., unpublished]

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 mutantwith PdGATL1.1

gatl1 mutantwith PdGATL1.2

T-DNAinsertiongatl1 mutantWild Type

EndogenousAtGATL1 gene

PdGATL1.1transgene

PdGATL1.1transcript

ACTIN

gatl1 mutants transformed with PdGATL1.2

WT gatl1 1 2 3 4 5 6T-DNA

insertion

EndogenousAtGATL1 gene

PdGATL1.2transgene

PdGATL1.2transcript

ACTIN

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

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

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

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]

0

5

10

15

20

25

30

35

40

45

GATL5 GATL6

rela

tive

fold

cha

nge

4 DPA7 DPA10 DPA

GATL5 and GATL6 transcript analysis in developing seed of Arabidopsis

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

Pectic polysaccharide

side-chain biosynthesis

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]

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 C

ell (2008) 20:1289-1302]

Pectin composition

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 esterificationof homogalacturonan (Mouille et al., 2007; Krupkova et al., 2007).

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

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

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