PBIO 691 9-24-2010
Yuning Chen
Department of Chemistry and
Biochemistry
PBIO 691 9-24-2010
Basic questions about something “NEW”:
1. What is it?
Definition, chemical composition, STRUCTURE, CHEMISTRY.
2. Where do I find it?
DISTRIBUTION in nature
3. Where does it come from/ where does it go?
Synthesis, trunover, METABOLISM
4. What does it do?
FUNCTION
5. What can I do with it?
APPLICATION, MANIPULATION
PBIO 691 9-24-2010
0. Hemicellulose
Definition by Encyclopaedia Britannica:
Hemicellulose, any of a group of complex carbohydrates that, with other carbohydrates
(e.g., pectins), surround the cellulose fibers of plant cells. The most common hemicelluloses
contain xylans (many molecules of the five-carbon sugar xylose linked together), a uronic
acid (i.e., sugar acid), and arabinose (another five-carbon sugar). Hemicelluloses have no
chemical relationship to cellulose.
More specific:
“A group of wall polysaccharides that are characterized by being neither cellulose nor
pectin and by having β-(1→4)- linked backbones of glucose, mannose, or xylose.”
Members of the hemicellulose family:
1.
2.
3.
4.
Xyloglucan (XyG): backbone made of β-(1→4)- glucans.
Xlylans : backbone made of β-(1→4)- linked xylose residues.
Mannans and Glucomannans: backbone contains mannose.
β-(1→3,1→4)-glucans.
"hemicellulose." Encyclopædia Britannica. 2010. Encyclopædia Britannica Online. 18
Sep. 2010 <http://www.britannica.com/EBchecked/topic/260780/hemicellulose>.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89
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1. Xyloglucan: distribution
Occurrence of hemicelluloses in primary and secondary walls of plants
XyG exists mainly in primary cell walls of land plants.
Indicates its function related to cell expansion and elongation.
XyG is the most abundant hemicellulose in primary walls of spermatophytes except for
grasses.
XyG structure varies among different plant tissues and species.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89.
PBIO 691 9-24-2010
2. Xyloglucan: structure
Sugars in XyG
Sugars in XyG backbone: Glucose (Glc)
Sugars in XyG branches:
Xylose (Xyl)
Arabinose (Ara)
Galactose (Gal)
Fucose (Fuc)
B. Buchanan, W. Gruissem, R. Jones, Eds. Biochemistry &
Molecular Biology of Plants, 2000, pp 57.
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2. Xyloglucan: structure
Sugar arrangement in XyG
Backbone: β-(1→4)-linked glucan chains.
Branch:
Reducing
glucose residues
converted to
alditol moieties
XA
XS
Xyloglucan nomenclature, CCRC website, accessed Sep 20, 2010.
<http://www.ccrc.uga.edu/~mao/xgnom/nomen.html>
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2. Xyloglucan: structure
Polysaccharide structure
(B/S/C)XXG
X X B G
Non commelinoids monocots and
most of the dicots.
X(F/J)(F/J)G
X L X G
X X G G
X X (F/J) G
X A G G
Solanaceous plants, Lamiales.
X A G G
L A G G
XXXGs contain Fuc on their side chain--- fucogalacto-XyGs.
XXGGs contain Ara on their side chain--- arabino-XyGs.
Vincken, J.P, York, W, S. et, al. Plant Physiol. 1997, 114, 9-13.
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2. Xyloglucan: structure
Polysaccharide structure
Possible forms of XXXG
Type
Source
XLLG
Storage
polysaccharides in seeds such as cyclamen…
XXFG/XLFG/XFFG
Primary cell wall (fucogalacto-XyG)
F replaced by J: α-L-Fucp-(1→2) replaced by α-LGalp-(1→2).
Arabidopsis fucose deficient mutant mur1.
(B/S/C)XXG
Sycamore.
XXBG
Sycamore.
Other types of XyG
Type
Source
XXG
Sycamore, apple
XXGGG
Immature barley plants, rice seedlings
XXXX
Seeds of Helipterum eximium
B. Buchanan, W. Gruissem, R. Jones, Eds. Biochemistry & Molecular
Biology of Plants, 2000, pp 65.
Vincken, J.P, York, W, S. et, al. Plant Physiol. 1997, 114, 9-13.
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2. Xyloglucan: structure
Methodology
1. XyG extraction: strong alkaline solutions, endoglucanase….
2. XyG digestion: endo-1,4-β-glucanases.
Type
Digestion product
(XXXG)n
XXXG.
(XXGG)n
Depends on the specificity
of the enzymes used.
3. Complete hydrolysis: strong acid.
4. Determination of sugar type: gas-liquid chromatography.
5. Determination of unit structure:
Methylation followed by MS.
NMR.
(a) Topographical image of xyloglucan deposited
onto mica from a 0.25 mg/mg sol diluted from
0.025% w/w. Image size is 2.5 mmx2.5 mm. (b)
The height profile of the cross section highlighted
in the image (a).
6. Morphology: Microscopy (TEM, AFM…).
B. Buchanan, W. Gruissem, R. Jones, Eds. Biochemistry &
Molecular Biology of Plants, 2000, pp 59-63.
Ikedaa, S, Nitta, Y. et.al. Food Hydrocolloids. 2004, 18,
669–675.
3. Xyloglucan: biosynthesis
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Step 1: obtaining of the starting material
https://www.msu.edu/~smithe44/calvin_cycle_process.htm,
accessed Sep, 21, 2010.
3. Xyloglucan: biosynthesis
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Step 2: generation of
individual building blocks
Overview of nucleotide sugar
interconversions relevant to the
synthesis of cell wall polymers in
higher plants.
Reiter, W, D. And Vanzin, Z, F. Plant Molecular
Biology. 2001, 47: 95–113.
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3. Xyloglucan: biosynthesis
Step 3: generation of the XyG backbone
The β-(1→4)- linked glucan backbone of XyG is generated by members of cellulose
synthase-like (CSL) proteins that belong to the CSLC family.
Schematic illustration of the phylogeny of the cellulose
synthase (CESA) and cellulose synthase–like (CSL)
superfamily of GTs. Not all families are present in the same
species. CSLH and CSLF are only known from grasses, and
CSLB and CSLG are only known from dicots. CSLE is known
from all angiosperms, but not outside these groups. CESA,
CSLA, CSLC, and CSLD are known from all plant genomes
analyzed so far. CSLJ is present in some angiosperms, both
dicots and grasses, but not in arabidopsis and rice.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89.
PBIO 691 9-24-2010
3. Xyloglucan: biosynthesis
Example case: Arabidopsis CSLC4 gene is involved in synthesizing the XyG backbone
Probe: partial arabidopsis MUR3 homolog
Maturation stage nasturtium seeds
1. The 5 CSLC genes were derived from the same gene.
2. That gene related to Arabidopsis CSLC4 with 77% identity and 85% similarity.
Express TmCSLC, AtCSLC4 and AtCSLC4/AtXT1 genes in yeast P. pastoris.
Product analysis
Insoluble
Soluble: from lines expressing AtCSLC4 alone
Gel-exclusion chromatography, HPAEC, permethylation
analysis, MALDI, NMR.
Linkage analysis of the oligosaccharide
content in the soluble fraction by gas
chromatography after permethylation.
Cocuron, J, C, Lerouxel, O. PNAS, 2007, 104, 8550-8555.
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3. Xyloglucan: biosynthesis
Step 4: generation of the side chains
Glycosyltranserases (GTs) involved in Xyloglucan biosynthesis
List of corresponding references:
24. Cocuron JC, Lerouxel O, et al. Proc. Natl. Acad. Sci. USA, 2007, 104, 8550–55.
34. Faik A, Price NJ, et al. Proc. Natl. Acad. Sci. USA, 2002, 99, 7797–7802.
79. Levy S, York WS, et al. Plant J. 1991, 1, 195–215.
84. Madson M, Dunand C, et al. Plant Cell, 2003, 15, 1662–1670.
106. Perrin RM, DeRocher AE, et al. Science, 1999, 284, 1976–1979.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89.
3. Xyloglucan: biosynthesis
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Step 5: modification
Acetylation:
1.
2.
3.
Mostly on O-6 of the galactose residues.
Occurs in the Golgi by means of transferases using acetyl-CoA.
None of the acetyltransferases or acetyl-CoA transporters required for this process have been identified.
Hydrolases:
Glycoside hydrolases (GHs) trim the nascent XyG chain, help keeping them soluble during transport and
incorporation into the wall, and more importantly, determine XyG structures in the wall.
In Arabidopsis, there are 730
open reading frames
corresponding to GHs and GTs,
of which 379 represent GHs
classed in 29 families.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89.
Minic, Z. Planta, 2008, 227, 723–740.
4. Xyloglucan: function
PBIO 691 9-24-2010
1. Binds to cellulose microfibers through non-covalent (H-Bonding) interactions, coats and cross-links
adjacent microfibers and serves as load-bearing glycans in primary cell wall.
Solution
conformation
XTHs, as polysaccharide
topoisomerase
Cellulose bindingfavored
conformation
2. Serves as a source of signal molecules:
XXFG counteracts auxin-induced cell expansion.
3. Serves as seed storage carbonhydrade.
http://www.ccrc.uga.edu/~mao/xyloglc/Xtext.htm, accessed Sep 22, 2010.
http://micro.magnet.fsu.edu/cells/plants/cellwall.html , accessed Sep 22, 2010.
Scheller, H, V and Ulvskov, P. Annu. Rev. Plant Biol. 2010. 61:263–89.
XTH family of enzymes, a brief view
Xyloglucan endo-Transglycosylase/Hydrolase (XTH):
1. Xyloglucan Endo-Transglycosylase (XET)
2. Xyloglucan Endo-Hydrolase (XEH)
Exons: open boxes, introns: lines, putative signal
peptide:the crosshatched boxes, catalytic site: filled
boxes.
Rose, J, Braam, J, et al. Plant Cell Physiol. 2002, 43, 1421–1435.
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XTH family of enzymes, a brief view
Working mechanism:
Active site: DEIDFEFLG or DEIDIEFLG
(1)
(2)
(3)
(1) Surface representation of PttXET1634 in silver (C-terminal extension in
copper) with XLLGXLLG
octadecasaccharide bound. (2) Cartoon of
PttXET16-34 showing the structure of the
C-terminal extension (copper) and the
catalytic amino acids (green) with a
bound XLLGXLLG octadecasaccharide.
(3)Overlay of PttXET16-34 (silver with red
loops), and an XEH, TmNXG1 (gold with
blue loops).
Mechanism
Substrate
Donor
Glc8- based XGOs
Acceptor
Glc4- based XGOs
Eklof, J, M and Brumer, H. Plant Physiol, 2010, 153, 456–466.
Rose, J, Braam, J, et al. Plant Cell Physiol. 2002, 43, 1421–1435.
XTH family of enzymes, a brief view
PBIO 691 9-24-2010
XTH actions in active primary cell walls
Cell wall assembly: integrate newly synthesized XyG pieces
into the cell wall network (MF: cellulose microfiber).
Cell expansion: enlarge space between
two cellulose microfibers.
Rose, J, Braam, J, et al. Plant Cell Physiol. 2002, 43, 1421–1435.
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Step #3
The Plant Cell, Vol. 20: 1519–1537, June 2008
How would arabidopsis cell wall be affected when it lacks XT genes?
What would happen chemically to the wall without XyGs?
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Step #1: Identification of AtXT1 (XXXT1).
UDP- [14C]Xyl
UDP- Glc
Pea membrane
[14C] XyG
Substrate for such enzymes are G5
Pea -Xylosyltransferase Shares Molecular Mechanisms
Similar to Fenugreek (1,6) Galactosyltransferase.
Arabidopsis data base for sequence similarities.
7 genes, including AtXT1, was identified
7 genes transformed to and expressed in yeast P.
pastoris, reacted with substrate and product identified.
AtXT1 was the α-Xylosyltransferase
Faik, A, Price, N, J, et al. PNAS, 2002, 99, 7797-7802.
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Step #1: Identification of AtXT2 (XXXT2).
AtXT2 was 83% identical and 91%
similar to AtXT1 at amino acid level
AtXT1 and AtXT2 were expressed
in insect cell lines.
Product analysis for both proteins
against G5 were similar.
Both proteins can add multiple Xyl
residues to G5.
Cavalier, D, M and Keegstra, K. JBC, 2006, 281, 34197–34207.
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Getting started: prepare the xxxt1, xxxt2 and
xxxt1 xxxt2 double knock-outs
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Observation: dwarfed plant abnormal root hair morphology
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What about XyGs?
The double knock-out lacked detectable XyG. Why?
1. OLIMP of the XEG digested cell wall
Conclusion: the double knock-outs
lacked detectable XyG?
NOT NOW
A HPAEC-PAD analysis for the double knock-out walls.
(B) Wild type.
(C) Double knock-outs
Caution:
1. OLIMP only analysis XyG that
spans adjacent cellulose
microfibers.
2. XEG wouldn’t be necessarily
able to reach this domain.
What about XyGs?
The double knock-out lacked detectable XyG. Why?
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2. Immunal labeling of the root cell wall polysaccharides
Caution:
XyG antibodies recognize
limited set of XyG epitops.
The labeling patterns (with
XyG specific antibodies)
between wild type and
single knock-outs might
differ slightly but there was
still indication of presence of
XyGs in those cell wall, while
the double knock-out cell
wall showed no labeling.
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2. Immunal labeling of the root cell wall polysaccharides
Xylan
Labeling all types of cell walls with antibodies that against other wall polysaccharides, result
showed that the other wall polysaccharides were not altered with deletion of XT gene/genes.
What about XyGs?
The double knock-out lacked detectable XyG. Why?
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3. Glycosyl residue composition and glycosyl linkage analysis
TFA hydrolysis liberates monosaccharides from noncellulosic polysaccharides and amorphous cellulose, while Saeman
hydrolysis releases sugars from crystalline cellulose and any remaining noncellulosic polysaccharides.
(1) The TFA treatment was exhaustive.
(2) There was a drop of Xyl level in the double knock-out.
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3. Glycosyl residue composition and glycosyl linkage analysis
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3. Glycosyl residue composition and glycosyl linkage analysis
There was a reduction in peak areas of glycosyl residues that can be assigned
to XyG, including T-Fuc, 2-Gal, 4-Glc, and 4,6-Glc with respect to the wild type
and the single mutants .
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3. Glycosyl residue composition and glycosyl linkage analysis
The xxt1 xxt2 double mutant has a significant reduction in the relative amounts of
glycosyl linkages that can be assigned to XyG.
What about XyGs?
The double knock-out lacked detectable XyG. Why?
4. Driselase hydrolysis
The absence of IP in double knock-out Driselase hydrolysis indicated there was no XyG
present in the double knock-out roots.
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What would be affected as a double knock-out?
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The xxxt2 knock-out and double knock-out affect the mechanical strength of the hypocotyls.
Conclusive remarks
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1. XXT1 and XXT2 encode XyG XTs that are required for XyG biosynthesis.
2. The xxt1 and xxt2 single mutants had a modest reduction in XyG and the xxt1 xxt2
double mutant lacks detectable XyG.
3. The reduction of XyG content in the xxt2 single mutant and the lack of detectable
XyG in the xxt1 xxt2 double mutant caused significant reductions in the stiffness and
ultimate strength parameters of these mutants.
Issues that remain unsolved
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1. Whether XXXT1 and XXXT2 genes are redundant?
2. How many genes are there that involves the production of the repetitive XXXGs?
3. Are XXXT1 and XXXT2 working with other genes? If so, who are those genes?
4. Without the supporting XyGs, how come the cell wall didn’t collapse?
5. From the cell wall point of view, how does it change morphologically after the
depletion of XyGs?