Matrix polysaccharide biosynthesis: Xyloglucans and Pectins

Transcription

1 Matrix polysaccharide biosynthesis: Xyloglucans and Pectins 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

2 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 ATP ADP GTP PPi L-Fuc 29 β-l-fuc-1-p 30 GDP-β-L-Fuc 27 GDP-4k-6d-D-Man GDP-L-Gul Ascorbate L-Gal L-Gal-1-P 40 GDP-L-Gal GDP-α-D-Man PPi GTP α-d-man-1-p mbp PPi UTP CoA AcCoA 43 ADP Glu ATP D-Man-6-P D-Man UDP-α-D-GlcNac GlcNac-1-P GlcN-6-P Gln UDP-Glc ADP ATP Frc6P Sucrose-6-P ADP-D-Gal D-Frc-6-P Frc Glc 32 Glc-1-P Gal-1-P Frc 45 ADP ATP 46 ADP-α-D-Glc D-Glc-6-P D-Glc TDP-β L-Rha TDP-D-Glc Frc PPi Frc ADP 44 4 TDP 3 2 ATP 3 GTP PPi 37 TDP-D-GlcA Sucrose α-d-glc-1-p GDP-D-Glc UDP 40 Pi 1 ATP ADP UTP PPi Frc GDP 3 13 UTP L-Rha β-l-rha-1-p UDP-β-L-Rha UDP-4k-6d-D-Glc PPi 9 ATP ADP UTP PPi UDP-α-D-Glc R-SO Glc6P Glc D-Gal α-d-gal-1-p UDP-α-D-Gal 8 3R NAD + 14 ATP ADP UTP PPi Ara-5-P PEP D-GalA α-d-gala-1-p UDP-α-D-GalA NADH UDP-α-D-sulfoQuin KDO-8-P O 2 ATP ADP UTP PPi 35 Glc6P Inositol D-GlcA α-d-glca-1-p UDP-α-D-GlcA CO 2 KDO 6 UTP PPi 5 CO 2 CTP Xyl α-d-xyl-1-p UDP-α-D-Xyl UDP-D-Api PPi 15 ATP ADP UTP PPi CMP-KDO L-Ara L-Ara-1-P UDP-L-Arap UDP-β-L-Araf The metabolism of NDP-sugars in plants: plant cell wall precursors. Mohnen, Bar-Peled and Somerville (2008).

5 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: ]

6 Mutation in GMD2 (mur1) results in reduced fucosylation in the non-meristematic regions of the root CCRC-M1 GMD1::GUS Freshour et al. (2003) Bonin et al. (2003)

7 Xyloglucan biosynthesis

8 Reverse genetic approaches have identified several genes encoding glycosyltransferases involved in xyloglucan biosynthesis CSLC FUT1 (MUR2) MUR3 XXT1 XXT2 XXT5 [Lerouxel et al., Curr. Opin. Plant Biol. (2006) 9: ]

9 Oligosaccharide content of wild-type and mur2 xyloglucans [Vanzin et al. (2002) Proc. Natl. Acad. Sci. 99: ]

10 Phylogenetic tree of putative xylosyl- and glycosyltransferases belonging to CaZY Family GT34 [Zabotina et al. (2008) Plant J. 56: ]

11 Localization of xyloglucan epitopes in wild-type and xylosyltransferase mutants of Arabidopsis [Cavalier et al. (2008) Plant Cell 20: ]

12 Oligosaccharide content of wild-type and mur3 xyloglucans [Madson et al. (2003) Plant Cell 15: ]

13 Pectin biosynthesis

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

15 Pectic polysaccharide backbone biosynthesis

16 Phylogenetic tree of CaZY Family GT8 indicates two distinct families of plant GT8 proteins Three major classes of GT8 proteins in nature Non-cell wall GT8 proteins in plants Cell wall-related GT8 proteins in plants [Yin et al., Plant Physiol. (2010) 153: ] A putative progenitor of cell wall-related GT8 genes in plants

17 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.

18 Comparative phylogenetic analysis of Arabidopsis thaliana, Populus trichocarpa and Oryza sativa GAUTs AtGAUT8 Qua1?HG:GalAT Os02g29530 AtGAUT8 eugene eugene fg4 pm.c LG XIII estext_gw1plus.c_ AtGAUT10 Os04g54360 B-2 grail eugene AtGAUT11 AtGAUT15 estext_gw1plus.c_lg_xiv2539 Os03g30000 AtGAUT9 eugene B-1 Os02g51130 Os06g12280 Os10g21890 Os06g51160 AtGAUT3 AtGAUT2 eugene Os06g49810 (60) A-1 AtGAUT1 HG:GalAT Os09g36190 Os09g36180 AtGAUT1 estext Gw1Plus.C LG XIV0504 eugene A-2 AtGAUT4 eugene fg4_pg.c_lg_vi Os08g23780 (79) AtGAUT7 A-4 Os09g30280 e gw1.xvi estext Gw1Plus.C Os08g38740 Os05g40720 Os12g02910 Os11g03160 AtGAUT7 AtGAUT12 Irx8 Os01g ?HG:GalAT or Xylan Primer/Cap? Os03g11330 (67) Os12g38930 AtGAUT14 AtGAUT13 eugene eugene AtGAUT12 estext fg4 pm.c LG XIII0357 eugene C Os11g37980 Os03g21250 eugene AtGAUT6 Os07g48370 AtGAUT5 fg4 pg.c LG II A-3 [Caffall et al. (2009) Molecular Plant 2: ]

19 GAUT1 & GAUT7 Predicted Type II transmembrane proteins Glycosyltransferase Family 8 (CAZy database - Predicted GT-A structure Golgi residents (Dunkley et al., 2004, 2006) GAUT1 (At3g61130) GAUT7 (At2g38650) Type II transmembrane protein (Keegstra and Raikhel Curr. Opin. Plant Biol. 4: ) 619 a.a kda pi a.a kda pi 9.95 N-terminal tail transmembrane domain stem globular / catalytic domain [from Debra Mohnen] (Topology prediction using HMMTOP v.2)

20 ExoPG Expt. Sensitivity of product produced by GAUT1- immunosorbed enzyme to expolygalacturonase proves synthesis of HG Water Boiled EPG EPG pmol GalA incorp. G Initial SP protein GAUT1 depleted GAUT1 enriched 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]

21 Heterologously expressed GAUT7 does not have HG:GalAT activity GAUT1 GAUT7 [Sterling et al. (2006) Proc. Natl. Acad. Sci. 103: ]

22 Mutation in GAUT8 (QUA1) affects morphology and cell adhesion [Bouton et al. Plant Cell (2002) 14: ]

23 Mutation in GAUT8 (QUA1) affects both GalA and Xyl content of Arabidopsis stem cell walls [Orfila et al. Planta (2005) 222: ]

24 Phylogenetic tree of the GATL family GATL-a GATL-b GATL-c GATL-g GATL-d GATL-f GATL-e [Kong et al., Plant Physiol. (2010), submitted ]

25 Pairs of GATL genes that are associated with genome duplication events in Arabidopsis Recent dupl. Older dupl. [Kong et al. Plant Physiol. (2010) submitted]

26 Plant growth and xylem/fiber morphology are altered in the gatl1/parvus/glz mutant A w.t. gatl1 Ph B Ve if Fiber Stem if Ph Xylem vessel Ve C Ve Xylary fiber D Hypcotyl Ve Vessel [Kong et al., unpublished]

27 Expression of PdGATL1.1 and PdGATL1.2 genes in gatl1/parvus/glz plants restores w.t. growth phenotypes gatl1 mutants transformed with PdGATL1.1 WT gatl Wild Type gatl1 mutant with PdGATL1.1 gatl1 mutant with PdGATL1.2 gatl1 mutant T-DNA insertion Endogenous AtGATL1 gene PdGATL1.1 transgene PdGATL1.1 transcript ACTIN T-DNA insertion gatl1 mutants transformed with PdGATL1.2 WT gatl Endogenous AtGATL1 gene PdGATL1.2 transgene PdGATL1.2 transcript ACTIN [Kong et al. (2009) Mol. Plant 2: ]

28 Expression of PdGATL1 genes in gatl1/parvus/glz plants mostly restores w.t. tissue morphology [Kong et al. (2009) Mol. Plant 2: ]

29 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]

30 GATL5 and GATL6 transcript analysis in developing seed of Arabidopsis 45 relative fold change DPA 7 DPA 10 DPA GATL5 GATL6 [Kong et al. (2010), in preparation]

31 Pectic polysaccharide side-chain biosynthesis

32 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]

33 Mutation in XGD1 results in reduced Xyl content in Arabidopsis pectin but not in xylan/xyloglucan Cell wall composition Pectin composition [Jensen et al. Plant Cell (2008) 20: ]

34 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).

35 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]