Bioenergy and the Plant Cell Wall

Transcription

1 Bioenergy and the Plant ell Wall

2 Understanding biomass biology is critical for biofuels production because ell wall architectures impact plant stature and form (biomass quantity) The architecture of cell walls limits their deconstruction to substrates for biofuel production (biomass quality) Plant cell walls may be optimized for their end-use in biofuel conversion processes (tailored biomass)

3 Primary vs. secondary cell walls Primary walls Middle lamella Pectin-rich cell corner

4 200 nm Mcann et al J. ell Sci. 96,

5 ellulose microfibrils are paracrystalline arrays of 24 to 36 chains of (1 4)-β-D-glucan

6 Sugar Structure and Nomenclature

7 Different sugars arise through epimerization and modification of primary alcohols β-l-arabinose (Arap) α-l-arabinose (Araf)

8 The enzymes of nucleotide interconversion are known UAM UDP L-Arap UDP L-Araf For all enzymes, UDP- and GDP-α-D- and β-l-sugars are made

9 Two observations about polysaccharide structures in plants: 1.Plants possess a small number of backbone polymers that, with few exceptions, are common to them all 2.Plants have an enormous diversity of side-group substitution patterns that impart specificity of function and physical properties in a species-specific manner* *This results in a large number of genes devoted to glycosyl transfer in a species-specific manner

10 ellulose microfibrils are tethered by cross-linking glycans (hemicelluloses) Fucogalacto-xyloglucan [ ] [ ] [ ] Arabino-xyloglucan Glucuronoarabinoxylan

11 Two types of uronic acid-rich pectins form a structurally interactive matrix around the cellulosic framework Homogalacturonans (HGs): Methyl esters Polygalacturonic acid (PGA) Xylogalacturonan

12 H Homogalacturonans may form "junction zones" with calcium H H H H H H H H H H H H H H H H H H H H H a 2+ a 2+ a 2+ a 2+ H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H a2+ H H H H H H H H H H H H H H H H a 2+ 3 a 2+ H 3 H H H H H 3 H H H H H H H H H H a 2+ H 3 H H H H H H H H H H H H H H H H H H H H

13 Rhamnogalacturonan II is one of the most complex biopolymers in nature H H H H 11 H H H 2H 21 H H HH H H3 HH 2 H H H H 9 H H 8 7 H H H 6 H H H 5 H H H H 4 3 H H H 2 H H 2 H H 10 H H H H H H H 2 H H H H H H H 2H H H H H H H H H H 19 H 2 H H H H

14 RG II, a complex pectic polysaccharide, may form di-diester cross-bridges with boron B

15 Rhamnogalacturonan I is the second type of pectin: it typically possesses arabinan and galactan sidegroups at the -4 position of Rha 5-arabinan 2-Rha 4-GalA 4-galactan Acetyl groups 2,4-Rha type I arabinogalactan

16 Networks of HG, RG I and its associated polysaccharides, and RG II form massive networks the constitute most of the interstitial space around the cellulose-glycan framework

17 Seed mucilages have altered RG-I structures Arabidopsis Flax Naran et al. (2008) Plant Physiology

18 Methylation analysis is a way to determine the linkage distribution H H H H H H 2 H H H H H H H 2 H H HH 2 H H H H H H H H 2 H Methylation with I H 2 H 2 H 3 H 3 H 3 H 3 H 3 H 2 H 2 Hydrolysis to monosaccharide Reduction with Na borodeuteride Acetylation

19 D H H H H H D H H H H H 2 D H H H H H 2 t-fucose t-xylose 2-Xylose D H H H H H H 2 D H H H H H H 2 D H H H H H H 2 2-Galactose 4-Glucose 4,6-Glucose

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21 In flax mucilage, the RG-I has t-fuc and t-l-gal at the -3 position, instead of arabinans and galactans at the -4 position; Arabidopsis also has small amounts of this special kind of RG-I in addition to the conventional kind 2,3-Rha 2,4-Rha

22 A composite of Arabinoxylan and RG I has enhanced viscosity

23 (Bruce Stone: This is the economy model) Model of a primary (growing) cell wall Mcann and Roberts, 1991

24 (Bruce Stone: This is the baroque model) The Type I wall arpita and Gibeaut,, 1993

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26 The easiest way to tell them apart is by the brilliant autofluorescence in UV light Arabidopsis leaf Maize leaf Micrographs by hris Staiger & Jessica Henty, Purdue Univ

27 β-glucans are unbranched polymers of cellodextrin oligomers connected by single (1 3)-linkages (1 3),(1 4)-β-D-glucan H H 2 H H HH 2 H H H H 2 H H H H H HH 2 H H H 2 H H H H HH 2 H H H 2 H HH HH 2 2 H 2 H H H H H H H H 2 H

28 The cell walls of grasses are unique among the angiosperms The Type II primary wall arpita and Mcann (2008) Trends Plant Sci 13: after arpita and Gibeaut (1993) Plant J. 3: 1-30

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30 The wall loosening enzymes are Expansins Xyloglucan endo-βtransglucosylases (XETs) can cut and ligate xyloglucan molecules

31 Xyloglucans are the principal cross-linking glycan of non-commelinoids XXXG XXFG ellotetraosyl unit backbone Three contiguously linked glucose units bear xylosyl units at the -6 Galactose may extend the side-chain at the middle xylose or first xylose; fucose may be added to the Gal of the first xylose to make a trisaccharide side-group. Trichoderma cellulase cleaves the polymer at the reducing end of the unbranched glucosyl residue

32 Side-group glycosylations function in xyloglucan dynamics during growth 6 Fuc 5 Gal (Ara) 7 Gal

33 Xyloglucan oligomers of defined structure are yielded by the Trichoderma cellulase ligomer Notation* [Molecular mass + Na+] Xyl Xyl Glc Glc Glc Glc Xyl Xyl Xyl Glc Glc Glc Glc Xyl Gal Gal Xyl Xyl Glc Glc Glc Glc Xyl Fuc Gal Xyl Xyl Glc Glc Glc Glc Xyl Gal Xyl Xyl Glc Glc Glc Glc Xyl Gal Fuc Gal Xyl Xyl Glc Glc Glc Glc Xyl Gal XXXG XLXG XXLG XXFG XLLG XLFG

34 Xyloglucan oligomeric units are separated by high performance anion exchange chromatography and detected by pulsed amperometry PAD Response XXXG XXFG XLFG XLXG Elution Time, min

35 Xyloglucan oligosaccharide composition can be determined by electrospray MS XXXG Relative Intensity XLXG + XXLG XXFG XLFG m/z

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37 Xyloglucans are predicted to adopt a twisted or flat conformation, and the side-groups may push towards flat

38 Two enzymes involved in the synthesis of fucogalacto-xyloglucans side-chains mur2 2 mur

39 All three enzymes that decorate fucogalacto-xyloglucans side- chains were identified by HPAE and ESI MS-MS of xyloglucans from mutants lacking these functional enzymes ESI MS-MS of m/z 1247 of mur3 xyloglucans shows lack of Gal at the first Xyl

40 Loss of tensile strength in the floral stem is associated only with the mur1 and mur1/mur2 double mutant Relative Tensile Strength1.25 wild-type mur1 mur1/ mur2 mur3 mur2

41 mur2 and mur3 exhibit altered xyloglucan oligomer patterns XXXG XXFG Wild-type XLFG XXXG XXLG XLLG mur2 In mur1 and mur2, XXLG and XLLG are major oligomers in addition to XXXG PAD Response XXXG XLXG XXLG XLXG mur1 XLLG + XXJG XXXG XLXG XLXG mur3 XXLG and XLLG are enhanced in abundance in mur2 In mur3, XLXG is the only oligomer in addition to XXXG, and its level is enhanced in leaves

42 We used a Texture Analyzer to determine the tensile strength of etiolated hypocotyls of Arabidopsis

43 Galactosylation is NT enhanced in etiolated hypocotyl, and the tensile strength of mur3 hypocotyls is severely compromised * *** wild-type mur2 mur3

44 Early growth of mur3 hypocotyls is normal, but considerable swelling occurs at the base after growth

45 There are small differences in the rates of cellulose binding in vitro, but tenacity of binding in vivo and in vitro are essentially the same wild-type mur2 mur3 y = x y = x y = x wt mur2 mur3 wt-rebound mur2-rebound mur3-rebound mur wild-type mur log 10 Time, min log 10 [NaH], mm

46 Further polymerization in muro of secreted XyG polymers is not compromised in mur2 or mur wild-type mur3 mur Fraction Number

47 The biggest difference was found in the activity of xyloglucan endo-β-transglucosylase towards low galactosylated xyloglucans A tamarind mur3 plants wild-type hypos mur2 cells Incorporation, d mur3 cells B tamarind wild-type mur2 mur [Xyloglucan], mg/ml Incubation Time, min Peña et al. (2004) Plant Physiology

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49 Expansins appear to be the major proteins involved in inducing wall stress relaxation, whereas XET activity religates xyloglucans with new neighbors during growth to maintain tensile strength

50 What does all this have to do with improving plant productivity for biomass? ell wall-related genes comprise about 10% of plant genomes A grand challenge is to define the gene networks that encode and coordinate the synthase complexes for specific wall architectures We also need to know how these synthase complexes work and the extent to which they can be tailored to produce new architectures