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Whole plant cell wall characterization using solution-state 2D NMR

Abstract

Recent advances in nuclear magnetic resonance (NMR) technology have made it possible to rapidly screen plant material and discern whole cell wall information without the need to deconstruct and fractionate the plant cell wall. This approach can be used to improve our understanding of the biology of cell wall structure and biosynthesis, and as a tool to select plant material for the most appropriate industrial applications. This is particularly true in an era when renewable materials are vital to the emerging bio-based economies. This protocol describes procedures for (i) the preparation and extraction of a biological plant tissue, (ii) solubilization strategies for plant material of varying composition and (iii) 2D NMR acquisition (for typically 15 min–5 h) and integration methods used to elucidate lignin subunit composition and lignin interunit linkage distribution, as well as cell wall polysaccharide profiling. Furthermore, we present data that demonstrate the utility of this new NMR whole cell wall characterization procedure with a variety of degradative methods traditionally used for cell wall compositional analysis.

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Figure 1: 2D NMR spectra revealing lignin unit compositions.
Figure 2: 2D NMR spectra revealing lignin interunit distributions, polysaccharide acylation (and polysaccharides).
Figure 3: 2D NMR spectra revealing polysaccharide anomerics.

References

  1. Weng, J.K. & Chapple, C. The origin and evolution of lignin biosynthesis. New Phytologist. 187, 273–285 (2010).

    CAS  Google Scholar 

  2. Vanholme, R., Morreel, K., Ralph, J. & Boerjan, W. Lignin biosynthesis and structure. Plant Physiol. 153, 895–905 (2010).

    PubMed  PubMed Central  CAS  Google Scholar 

  3. Ralph, J. et al. Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Revs. 3, 29–60 (2004).

    CAS  Google Scholar 

  4. Boerjan, W., Ralph, J. & Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Biol. 54, 519–549 (2003).

    PubMed  CAS  Google Scholar 

  5. Sarkanen, K.V. & Ludwig, C.H. Lignins, Occurrence, Formation, Structure and Reactions (Wiley-Interscience, 1971).

  6. Freudenberg, K. & Neish, A.C. Constitution and Biosynthesis of Lignin (Springer-Verlag, 1968).

  7. Ralph, J. & Landucci, L.L. NMR of lignins. in Lignin and Lignans; Advances in Chemistry (eds. Heitner, C., Dimmel, D.R. & Schmidt, J.A.) 137–234 (CRC Press, 2010).

  8. Ralph, J. Hydroxycinnamates in lignification. Phytochem. Revs. 9, 65–83 (2010).

    CAS  Google Scholar 

  9. Vanholme, R., Morreel, K., Ralph, J. & Boerjan, W. Lignin engineering. Curr. Opin. Plant Biol. 11, 278–285 (2008).

    PubMed  CAS  Google Scholar 

  10. Ralph, J. et al. in Recent Advances in Polyphenol Research Vol. 1 (eds. Daayf, F., El Hadrami, A., Adam, L. & Balance, G.M.) Ch. 2, 36–66 (Wiley-Blackwell Publishing, 2008).

    Google Scholar 

  11. Ralph, J. Perturbing lignification. in The Compromised Wood Workshop 2007 (eds. Entwistle, K., Harris, P.J. & Walker, J.) 85–112 (Wood Technology Research Centre, University of Canterbury, 2007).

  12. Ralph, J. et al. Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls. Phytochem. Revs. 3, 79–96 (2004).

    CAS  Google Scholar 

  13. Ralph, J. et al. Cell wall cross-linking in grasses by ferulates and diferulates. et al. in Lignin and Lignan Biosynthesis, Vol. 697. (eds. Lewis, N.G. & Sarkanen, S.) 209–236 (American Chemical Society, 1998).

  14. Jung, H.G. & Allen, M.S. Characteristics of plant cell walls affecting intake and digestibility of forages by ruminants. J. Animal Sci. 73, 2774–2790 (1995).

    CAS  Google Scholar 

  15. Jung, H.G. Forage lignins and their effects on fiber digestibility. Agron. J. 81, 33–38 (1989).

    CAS  Google Scholar 

  16. Stewart, J.J., Kadla, J.F. & Mansfield, S.D. The influence of lignin chemistry and ultrastructure on the pulping efficiency of clonal aspen (Populus tremuloides Michx.) Holzforschung 60, 111–122 (2006).

    CAS  Google Scholar 

  17. Chen, F. & Dixon, R.A. Genetic manipulation of lignin biosynthesis to improve biomass characteristics for agro-industrial processes. In Vitro Cell. Dev. Biol.—Animal 44, S28–S29 (2008).

    Google Scholar 

  18. Li, X., Weng, J.K. & Chapple, C. Improvement of biomass through lignin modification. Plant J. 54, 569–581 (2008).

    PubMed  CAS  Google Scholar 

  19. Chapple, C., Ladisch, M. & Meilan, R. Loosening lignin's grip on biofuel production. Nat. Biotechnol. 25, 746–748 (2007).

    PubMed  CAS  Google Scholar 

  20. Bonawitz, N.D. & Chapple, C. The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu. Rev. Genet. 44, 337–363 (2010).

    PubMed  CAS  Google Scholar 

  21. Simmons, B.A., Loqué, D. & Ralph, J. Advances in modifying lignin for enhanced biofuel production. Curr. Opin. Plant Biol. 13, 313–320 (2010).

    PubMed  CAS  Google Scholar 

  22. Jouanin, L. et al. Comparison of the consequences on lignin content and structure of COMT and CAD downregulation in poplar and Arabidobsis thaliana. in Plantation Forest Biotechnology in the 21st Century (eds. Walter, C. & Carson, M.) 219–229 (Research Signpost, 2004).

  23. Boerjan, W. et al. in Molecular Breeding of Woody Plants, Vol. Progress in Biotechnology Series, Vol. 18 (eds. Morohoshi, N. & Komamine, A.), Ch. 23, 187–194 (Elsevier Science, 2001).

  24. Mansfield, S.D. Solutions for dissolution-engineering cell walls for deconstruction. Curr. Opin. Biotechnol. 20, 286–294 (2009).

    PubMed  CAS  Google Scholar 

  25. Lin, S.Y. & Dence, C.W. Methods in Lignin Chemistry (Springer-Verlag, 1992).

  26. Lapierre, C. Application of new methods for the investigation of lignin structure. in Forage Cell Wall Structure and Digestibility (eds. Jung, H.G., Buxton, D.R., Hatfield, R.D. & Ralph, J.) 133–166 (American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, 1993).

  27. Rolando, C., Monties, B. & Lapierre, C. Thioacidolysis. in Methods in Lignin Chemistry (eds. Dence, C.W. & Lin, S.Y.) 334–349 (Springer-Verlag, 1992).

  28. Robinson, A.R. & Mansfield, S.D. Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling. Plant J. 58, 706–714 (2009).

    PubMed  CAS  Google Scholar 

  29. Yamamura, M., Hattori, T., Suzuki, S., Shibata, D. & Umezawa, T. Microscale alkaline nitrobenzene oxidation method for high-throughput determination of lignin aromatic components. Plant Biotechnol. 27, 305–310 (2010).

    CAS  Google Scholar 

  30. Villar, J.C., Caperos, A. & GarciaOchoa, F. Oxidation of hardwood kraft-lignin to phenolic derivatives. Nitrobenzene and copper oxide as oxidants. J. Wood Chem. Technol. 17, 259–285 (1997).

    CAS  Google Scholar 

  31. Lu, F. & Ralph, J. Efficient ether cleavage in lignins: the derivatization followed by reductive cleavage procedure as a basis for new analytical methods. in Lignin and Lignan Biosynthesis (eds. Lewis, N.G. & Sarkanen, S.) 294–322 (American Chemical Society, 1998).

  32. Lu, F. & Ralph, J. Derivatization followed by reductive cleavage (DFRC method), a new method for lignin analysis: protocol for analysis of DFRC monomers. J. Agr. Food Chem. 45, 2590–2592 (1997).

    CAS  Google Scholar 

  33. Lu, F. & Ralph, J. The DFRC method for lignin analysis. Part 1. A new method for β-aryl ether cleavage: lignin model studies. J. Agr. Food Chem. 45, 4655–4660 (1997).

    CAS  Google Scholar 

  34. Morrison, I.M. Improvements in the acetyl bromide technique to determine lignin and digestibility and its application to legumes. J. Sci. Food Agr. 23, 1463–1469 (1972).

    CAS  Google Scholar 

  35. Morrison, I.M. Semimicro method for the determination of lignin and its use in predicting the digestibility of forage crops. J. Sci. Food Agr. 23, 455–463 (1972).

    CAS  Google Scholar 

  36. Fukushima, R.S. & Hatfield, R. Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples. J. Agr. Food Chem. 52, 3713–3720 (2004).

    CAS  Google Scholar 

  37. Fukushima, R.S. & Hatfield, R.D. Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method. J. Agr. Food Chem. 49, 3133–3139 (2001).

    CAS  Google Scholar 

  38. Yelle, D.J., Wei, D., Ralph, J. & Hammel, K.E. Multidimensional NMR analysis reveals truncated lignin structures in wood decayed by the brown rot basidiomycete Postia placenta. Appl. Environ. Microbiol. 13, 1091–1100 (2011).

    CAS  Google Scholar 

  39. Rencoret, J. et al. Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiol. 155, 667–682 (2011).

    PubMed  CAS  Google Scholar 

  40. Lu, F. & Ralph, J. Solution-state NMR of lignocellulosic biomass. J. Biobased Mater. Bio. 5, 169–180 (2011).

    CAS  Google Scholar 

  41. Kim, H. & Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5 . Org. Biomol. Chem. 8, 576–591 (2010).

    PubMed  CAS  Google Scholar 

  42. Hedenström, M. et al. Identification of lignin and polysaccharide modifications in Populus pood by chemometric analysis of 2D NMR spectra from dissolved cell walls. Mol. Plant 2, 933–942 (2009).

    PubMed  Google Scholar 

  43. Yelle, D.J., Ralph, J. & Frihart, C.R. Characterization of non-derivatized plant cell walls using high-resolution solution-state NMR spectroscopy. Magn. Reson. Chem. 46, 508–517 (2008).

    PubMed  PubMed Central  CAS  Google Scholar 

  44. Kim, H., Ralph, J. & Akiyama, T. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6 . BioEnergy Res. 1, 56–66 (2008).

    Google Scholar 

  45. Ralph, J. & Lu, F. Cryoprobe 3D NMR of acetylated ball-milled pine cell walls. Org. Biomol. Chem. 2, 2714–2715 (2004).

    PubMed  CAS  Google Scholar 

  46. Lu, F. & Ralph, J. Non-degradative dissolution and acetylation of ball-milled plant cell walls; high-resolution solution-state NMR. Plant J. 35, 535–544 (2003).

    PubMed  CAS  Google Scholar 

  47. Ralph, J. et al. Solution-state NMR of lignins. in Advances in Lignocellulosics Characterization (ed. Argyropoulos, D.S.) 55–108 (TAPPI Press, 1999).

  48. Bradley, S.A. & Krishnamurthy, K. A modified CRISIS-HSQC for band-selective IMPRESS. Magn. Reson. Chem. 43, 117–123 (2005).

    PubMed  CAS  Google Scholar 

  49. Boyer, R.D., Johnson, R. & Krishnamurthy, K. Compensation of refocusing inefficiency with synchronized inversion sweep (CRISIS) in multiplicity-edited HSQC. J. Magn. Reson. 165, 253–259 (2003).

    PubMed  CAS  Google Scholar 

  50. Zhang, S.M., Wu, J. & Gorenstein, D.G. 'Double-WURST' decoupling for N-15- and C-13-double-labeled proteins in a high magnetic field. J. Magn. Reson. Series A 123, 181–187 (1996).

    CAS  Google Scholar 

  51. Kupce, E. & Freeman, R. Compensation for spin-spin coupling effects during adiabatic pulses. J. Magn. Reson. 127, 36–48 (1997).

    CAS  Google Scholar 

  52. Ralph, S.A., Landucci, L.L. & Ralph, J. NMR Database of Lignin and Cell Wall Model Compounds. http://ars.usda.gov/Services/docs.htm?docid=10491 (2004).

  53. Brennan, M., McLean, J.P., Altaner, C., Ralph, J. & Harris, P.J. Cellulose microfibril angles and cell-wall polymers in different wood types of Pinus radiata. Cellulose 19, 1385–1404 (2012).

    CAS  Google Scholar 

  54. Weng, J.-K., Akiyama, T., Ralph, J., Golden, B.L. & Chapple, C. Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii. Plant Cell 23, 2708–2724 (2011).

    PubMed  PubMed Central  CAS  Google Scholar 

  55. Weng, J.-K. et al. Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants. Plant Cell 22, 1033–1045 (2010).

    PubMed  PubMed Central  CAS  Google Scholar 

  56. Vanholme, R. et al. Engineering traditional monolignols out of lignins by concomitant up-regulation F5H1 and down-regulation of COMT in Arabidopsis. Plant J. 64, 885–897 (2010).

    PubMed  CAS  Google Scholar 

  57. Stewart, J.J., Akiyama, T., Chapple, C.C.S., Ralph, J. & Mansfield, S.D. The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol. 150, 621–635 (2009).

    PubMed  PubMed Central  CAS  Google Scholar 

  58. Ralph, J. et al. Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl-CoA reductase deficiency). Plant J. 53, 368–379 (2008).

    PubMed  CAS  Google Scholar 

  59. Leplé, J.-C. et al. Downregulation of cinnamoyl coenzyme A reductase in poplar; multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell 19, 3669–3691 (2007).

    PubMed  PubMed Central  Google Scholar 

  60. Ralph, J. et al. Effects of coumarate-3-hydroxylase downregulation on lignin structure. J. Biol. Chem. 281, 8843–8853 (2006).

    PubMed  CAS  Google Scholar 

  61. Bunzel, M. & Ralph, J. NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber. J. Agr. Food Chem. 54, 8352–8361 (2006).

    CAS  Google Scholar 

  62. Marita, J.M., Vermerris, W., Ralph, J. & Hatfield, R.D. Variations in the cell wall composition of maize brown midrib mutants. J. Agr. Food Chem. 51, 1313–1321 (2003).

    CAS  Google Scholar 

  63. Goujon, T. et al. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Mol. Biol. 51, 973–989 (2003).

    PubMed  CAS  Google Scholar 

  64. Ralph, J. et al. Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochem. 57, 993–1003 (2001).

    CAS  Google Scholar 

  65. Marita, J., Ralph, J., Hatfield, R.D. & Chapple, C. NMR characterization of lignins in Arabidopsis altered in the activity of ferulate-5-hydroxylase. Proc. Natl. Acad. Sci. USA 96, 12328–12332 (1999).

    PubMed  PubMed Central  CAS  Google Scholar 

  66. Hu, W.-J. et al. Repression of lignin biosynthesis in transgenic trees promotes cellulose accumulation and growth. Nat. Biotechnol. 17, 808–812 (1999).

    PubMed  CAS  Google Scholar 

  67. Ralph, J., Akiyama, T., Coleman, H.D. & Mansfield, S.D. Effects on lignin structure of coumarate 3-hydroxylase downregulation in Poplar. BioEnergy Research, published online, doi:10.1007/s12155-012-9218-y (24 May 2012).

  68. Wagner, A. et al. Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata. Proc. Natl. Acad. Sci. USA 104, 11856–11861 (2007).

    PubMed  PubMed Central  CAS  Google Scholar 

  69. Zhang, L.M. & Gellerstedt, G. Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references. Magn. Reson. Chem. 45, 37–45 (2007).

    PubMed  CAS  Google Scholar 

  70. Heikkinen, S., Toikka, M.M., Karhunen, P.T. & Kilpeläinen, I.A. Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin. J. Am. Chem. Soc. 125, 4362–4367 (2003).

    PubMed  CAS  Google Scholar 

  71. Hatfield, R.D., Marita, J.M. & Frost, K. Characterization of p-coumarate accumulation, p-coumaroyl transferase, and cell wall changes during the development of corn stems. J. Sci. Food Agr. 88, 2529–2537 (2008).

    CAS  Google Scholar 

  72. Hatfield, R.D. et al. Grass lignin acylation: p-coumaroyl transferase activity and cell wall characteristics of C3 and C4 grasses. Planta 229, 1253–1267 (2009).

    PubMed  CAS  Google Scholar 

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Acknowledgements

We gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada′s Discovery Program held by S.D.M.; H.K., F.L., S.D.M. and J.R. were funded in part by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494).

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S.D.M., J.R., H.K. and F.L. conceived and designed the experiments. H.K. and F.L. performed the NMR experimental evaluation. J.R. contributed to spectral interpretation. S.D.M. performed the wet chemical evaluation of the cell walls. S.D.M. and J.R. wrote the paper (with input from H.K. and F.L.).

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Correspondence to Shawn D Mansfield or John Ralph.

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Mansfield, S., Kim, H., Lu, F. et al. Whole plant cell wall characterization using solution-state 2D NMR. Nat Protoc 7, 1579–1589 (2012). https://doi.org/10.1038/nprot.2012.064

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