Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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]