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XYLOGLUCAN 木葡聚糖

PBIO 691 9-24-2010. XYLOGLUCAN 木葡聚糖. 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.

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XYLOGLUCAN 木葡聚糖

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  1. PBIO 691 9-24-2010 XYLOGLUCAN 木葡聚糖 Yuning Chen Department of Chemistry and Biochemistry

  2. 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

  3. 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: 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

  4. PBIO 691 9-24-2010 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.

  5. PBIO 691 9-24-2010 2. Xyloglucan: structure Sugars in XyG Sugars in XyG backbone: Glucose (Glc) Sugars in XyG branches: Xylose (Xyl) Galactose (Gal) Arabinose (Ara) Fucose (Fuc) B. Buchanan, W. Gruissem, R. Jones, Eds. Biochemistry & Molecular Biology of Plants, 2000, pp 57.

  6. PBIO 691 9-24-2010 2. Xyloglucan: structure Sugar arrangement in XyG Backbone: β-(1→4)-linked glucan chains. Branch: Reducing glucose residues converted to alditol moieties A • X S X Xyloglucan nomenclature, CCRC website, accessed Sep 20, 2010. <http://www.ccrc.uga.edu/~mao/xgnom/nomen.html>

  7. PBIO 691 9-24-2010 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 (F/J) G X X G 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.

  8. PBIO 691 9-24-2010 2. Xyloglucan: structure Polysaccharide structure Possible forms of XXXG Other types of XyG 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.

  9. PBIO 691 9-24-2010 2. Xyloglucan: structure Methodology 1. XyG extraction: strong alkaline solutions, endoglucanase…. 2. XyG digestion: endo-1,4-β-glucanases. 3. Complete hydrolysis: strong acid. 4. Determination of sugar type: gas-liquid chromatography. (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). 5. Determination of unit structure: Methylation followed by MS. NMR. 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.

  10. PBIO 691 9-24-2010 3. Xyloglucan: biosynthesis Step 1: obtaining of the starting material https://www.msu.edu/~smithe44/calvin_cycle_process.htm, accessed Sep, 21, 2010.

  11. PBIO 691 9-24-2010 3. Xyloglucan: biosynthesis 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.

  12. PBIO 691 9-24-2010 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.

  13. 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. Product analysis Express TmCSLC, AtCSLC4 and AtCSLC4/AtXT1 genes in yeast P. pastoris. 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.

  14. PBIO 691 9-24-2010 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.

  15. PBIO 691 9-24-2010 3. Xyloglucan: biosynthesis Step 5: modification Acetylation: 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.

  16. PBIO 691 9-24-2010 4. Xyloglucan: function 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 binding-favored 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.

  17. PBIO 691 9-24-2010 XTH family of enzymes, a brief view Xyloglucanendo-Transglycosylase/Hydrolase (XTH): Xyloglucan Endo-Transglycosylase (XET) 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.

  18. PBIO 691 9-24-2010 XTH family of enzymes, a brief view Working mechanism: Active site: DEIDFEFLG or DEIDIEFLG (1) (2) (3) (1) Surface representation of PttXET16-34 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). 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.

  19. PBIO 691 9-24-2010 XTH family of enzymes, a brief view 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.

  20. PBIO 691 9-24-2010 Step #3 Disrupting Two Arabidopsis thaliana Xylosyltransferase Genes Results in Plants Deficient in Xyloglucan, a Major Primary Cell Wall Component. 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?

  21. PBIO 691 9-24-2010 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.

  22. PBIO 691 9-24-2010 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.

  23. PBIO 691 9-24-2010 Getting started: prepare the xxxt1, xxxt2 and xxxt1 xxxt2 double knock-outs

  24. PBIO 691 9-24-2010 Observation: dwarfed plant abnormal root hair morphology

  25. PBIO 691 9-24-2010 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 Caution: OLIMP only analysis XyG that spans adjacent cellulose microfibers. XEG wouldn’t be necessarily able to reach this domain. A HPAEC-PAD analysis for the double knock-out walls. (B) Wild type. (C) Double knock-outs

  26. PBIO 691 9-24-2010 What about XyGs? The double knock-out lacked detectable XyG. Why? 2. Immunal labeling of the root cell wall polysaccharides Caution: XyG antibodies recognize limited set of XyGepitops. 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.

  27. PBIO 691 9-24-2010 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.

  28. PBIO 691 9-24-2010 What about XyGs? The double knock-out lacked detectable XyG. Why? 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. The TFA treatment was exhaustive. There was a drop of Xyl level in the double knock-out.

  29. PBIO 691 9-24-2010 3. Glycosyl residue composition and glycosyl linkage analysis

  30. PBIO 691 9-24-2010 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 .

  31. PBIO 691 9-24-2010 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.

  32. PBIO 691 9-24-2010 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.

  33. PBIO 691 9-24-2010 What would be affected as a double knock-out? The xxxt2 knock-out and double knock-out affect the mechanical strength of the hypocotyls.

  34. PBIO 691 9-24-2010 Conclusive remarks 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.

  35. PBIO 691 9-24-2010 Issues that remain unsolved Whether XXXT1 and XXXT2 genes are redundant? How many genes are there that involves the production of the repetitive XXXGs? Are XXXT1 and XXXT2 working with other genes? If so, who are those genes? Without the supporting XyGs, how come the cell wall didn’t collapse? From the cell wall point of view, how does it change morphologically after the depletion of XyGs?

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