, Volume 201, Issue 3, pp 311–318 | Cite as

Fourier-transform infrared and Raman spectroscopic evidence for the incorporation of cinnamaldehydes into the lignin of transgenic tobacco (Nicotiana tabacum L.) plants with reduced expression of cinnamyl alcohol dehydrogenase

  • Derek Stewart
  • Nabila Yahiaoui
  • Gordon J. McDougall
  • Kate Myton
  • Christiané Marque
  • Alain M. Boudet
  • James Haigh


Xylem from stems of genetically manipulated tobacco plants which had had cinnamyl alcohol dehydrogenase (CAD; EC activity down-regulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly higher, lignin contents than xylem from wild-type plants. Fourier-transform infrared (FT-IR) microspectroscopy indicated that down-regulation of CAD had resulted in the incorporation of moieties with conjugated carbonyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lignin had altered. The FT-Raman spectra of manipulated xylem exhibited maxima consistent with the presence of elevated levels of aldehydic groups conjugated to a carbon-carbon double bond and a guaiacyl ring. These maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their absolute frequencies matched those of coniferaldehyde. Furthermore, xylem from clone 37 was found to have a higher content of carbonyl groups than that of clone 49 or the wild-type (clone 37: clone 49: wild-type; 2.4:1.6:1.0) as measured by a degradative chemical method. This is the first report of the combined use of FT-IR and FT-Raman spectroscopies to study lignin structure in situ. These analyses provide strong evidence for the incorporation of cinnamaldehyde groups into the lignin of transgenic plants with down-regulated CAD expression. In addition, these non-destructive analyses also suggest that the plants transformed with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type.

Key words

Lignin-Cell walls Cinnamyl alcohol dehydrogenase Infrared Raman 



cinnamyl alcohol dehydrogenase


Fourier-transform infrared


diffuse reflectance Fourier-transform infrared


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Agarwal UP, Atalla RH (1986) In-situ Raman microprobe studies of plant cell walls: macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P. Planta 169: 325–332Google Scholar
  2. Agarwal UP, Atalla RH (1994) Raman spectral features associated with chromophores in high-yield pulps. J. Wood Chem Technol 14: 227–241CrossRefGoogle Scholar
  3. Agarwal UP, Atalla RH, Forsskåhl I. (1995) Sequential treatment of mechanical and chemi-mechanical pulps with light and heat: a Raman spectroscopic study. Holzforschung 49: 300–312Google Scholar
  4. Akin DE, Himmelsbach DS, Carl RT, Hanna WW, Barton II FE (1993) Mid-infrared microspectroscopy to assess lignin in plant tissues related to digestibility. Agron J 85: 171–175CrossRefGoogle Scholar
  5. Atanassova R, Favet N, Maitz F, Chabbert B, Tollier MT, Monties B, Fritig B, Legrand M (1995) Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation. Plant J 8: 465–477CrossRefGoogle Scholar
  6. Bond JS, Agarwal UP, Atalla RH (1990) Contributions of preresonance Raman and conjugative effects to the Raman spectrum of native lignin. In: Durig JF, Sullivan JF (eds) The Twelfth International Conference on Raman Spectroscopy. South Carolina, USA, pp 652–653Google Scholar
  7. Boudet AM, Lapierre C, Grima-Pettenari J (1995) Biochemistry and molecular biology of lignification. New Phytol 129: 203–236CrossRefGoogle Scholar
  8. Campbell MM, Sederoff RS (1996) Variation in lignin content and composition. Mechanisms of control and implications for the genetic improvement of plants. Plant Physiol 110: 3–13PubMedGoogle Scholar
  9. Cui F, Dolphin D (1991) Iron porphyrin catalyzed oxidation of lignin model compounds: the oxidation of veratryl alcohol and veratryl acetate. Can J Chem 70: 2314–2318CrossRefGoogle Scholar
  10. Dean JFD, Eriksson KEL (1992) Biotechnological modification of lignin structure and composition in forest trees. Holzforschung 46: 135–147Google Scholar
  11. van Doorsselaere JV, Baucher M, Chogrot E, Chabbert B, Tollier MT, Petit Conil M, Leplé JC, Pilate G, Cornu D, Monties B, Van Montagu M, Inzé D, Boerjan W, Jouanin L (1995) A novel lignin in poplar with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity. Plant J 8: 855–864Google Scholar
  12. Dwivedi UN, Campbell WH, Yu J, Dalta RSS, Bugos RC, Chiang VL, Podila GK (1993) Modification of lignin biosynthesis in transgenic tobacco through expression of an antisense O-methyltransferase gene from Populus. Plant Mol Biol 26: 61–71CrossRefGoogle Scholar
  13. Ede RM, Brunow G, Simola LK, Lemmetyinen J (1990) Twodimensional 1H-1H chemical shift correlation and J-resolved NMR studies on isolated and synthetic lignins. Holzforschung 44: 95–101Google Scholar
  14. Elkind Y, Edwards R, Mavandad M, Hendrick SA, Dixon RA, Lamb CJ (1990) Abnormal plant development and downregulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonialyase gene. Proc Natl Acad Sci 87: 9057–9061PubMedCrossRefGoogle Scholar
  15. Faix O (1991) Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 45 [Suppl]: 21–27Google Scholar
  16. Gellerstedt G, Pettersson EL (1975) Light-induced oxidation of lignin. Part 2. The oxidative degradation of aromatic rings. Svensk Papperstidn 80: 15–21Google Scholar
  17. Goffner D, Campbell MM, Campargue C, Clastre M, Borderies G, Boudet A, Boudet AM (1994) Purification and characterization of cinnamoyl-CoA: NADP oxidoreductase in Eucalyptus gunnii. Plant Physiol 106: 625–632PubMedGoogle Scholar
  18. Halpin C, Knight ME, Grima-Pettenari J, Goeffner D, Boudet AM, Schuch W (1992) Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems. Plant Physiol 98: 12–16PubMedCrossRefGoogle Scholar
  19. Halpin C, Knight ME, Foxon GA, Campbell MM, Boudet AM, Boon JJ, Chabbert B, Tollier MT, Schuch W (1994) Manipulation of lignin quality by down regulation of cinnamyl alcohol dehydrogenase. Plant J 6: 339–350CrossRefGoogle Scholar
  20. Hergert HL (1969) Infrared spectra of lignin and related compounds. II. Conifer lignin and model compounds. J Org Chem 25: 405–413CrossRefGoogle Scholar
  21. Hergert HL (1970) Infrared spectra. In: Sarkanen KV, Ludwig CH (eds) Lignins; occurrence, formation, structure and reactions. Wiley Interscience, New York, pp 267–297Google Scholar
  22. Hibino T, Takabe K, Kawazu T, Shibata D, Higuchi T (1995) Increase of cinnamaldehyde groups in lignin of transgenic plants carrying an antisense gene for cinnamyl alcohol dehydrogenase. Biosci Biotech Biochem 59: 929–931Google Scholar
  23. Higuchi T (1985) Biosynthesis of lignin. In: Kratzl K, Billek G (eds) Biosynthesis and biodégradation of wood components. Academic Press, London, pp 141–160Google Scholar
  24. Higuchi T, Ito T, Umezawa T, Hibino T, Shibata D (1994) Redbrown colour of lignified tissues of transgenic plants with antisense CAD gene: Wine-red lignin from coniferaldehyde. J Biotechnol 37: 151–158CrossRefGoogle Scholar
  25. Himmelsbach DS, Barton II FE (1980) 13C Nuclear magnetic resonance spectroscopy of grass lignins. J Agric Food Chem 28: 1203–1208CrossRefGoogle Scholar
  26. Jouanin L (1995) Modification of lignin quality in poplar trees using antisense strategy. Polyphenols Actualites 13: 5–7Google Scholar
  27. Jung HJG, Deetz DA (1993) Cell wall lignification and degradability. In: Jung HG, Buxton DR, Hatfield RD, Ralph J (eds) Forage cell wall structure and digestibility, ASA-CSSA-SSSA, Madison, pp 315–346Google Scholar
  28. Kemp W (1991) Organic spectroscopy. Macmillan, London, pp 19–100Google Scholar
  29. Lewis NG, Newman J, Just G, Ripmeister J (1987) Determination of bonding patterns of 13C specifically enriched dehydrogenatively polymerised lignin in solution and solid state. Macromolecules 20: 1752–1756CrossRefGoogle Scholar
  30. Lundquist K (1991) 1H NMR spectral studies of lignins. Nordic Pulp Paper Res J 3: 140–146CrossRefGoogle Scholar
  31. McDougall GJ, Morrison IM, Stewart D, Weyers JDB, Hillman JR (1993) Plant fibres: botany, chemistry and processing for industrial use. J Sci Food Agric 62: 1–20CrossRefGoogle Scholar
  32. Morrison IM (1972) A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops. J Sci Food Agric 23: 455–463PubMedCrossRefGoogle Scholar
  33. Ni W, Paiva NL, Dixon RA (1994) Reduced lignin in transgenic plants containing a caffeic acid O-methytransferase antisense gene. Transgenic Res 3: 120–126CrossRefGoogle Scholar
  34. Pillonel C, Mulder MM, Boon JJ, Forster B, Binder A (1991) Involvement of cinnamyl alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench. Planta 185: 538–544Google Scholar
  35. Sarkanen KV, Chang HM, Ericsson B (1967) Species variations in lignins. Part 1. Infrared spectra of guaiacyl and syringyl models. Tappi 50: 572–575Google Scholar
  36. Sederoff R, Campbell M, O’Malley D, Whetten R (1994) Genetic regulation of lignin biosynthesis and the potential modification of wood by genetic engineering in loblolly pine. In: Ellis B (ed) Genetic engineering of plant secondary metabolism. Plenum Press, New York, pp 313–355Google Scholar
  37. Stewart D, Morrison IM (1992) FT-IR spectroscopy as a tool for the study of biological and chemical treatments of barley straw. J Sci Food Agric 60: 431–436CrossRefGoogle Scholar
  38. Stewart D, Wilson HM, Hendra PJ, Morrison IM (1995) Fouriertransform infrared and Raman spectroscopic study of biochemical and chemical treatments of oak (Quercus rubra) wood and barley (Hordeum vulgare) straw. J Food Agric Chem 43: 2219–2225CrossRefGoogle Scholar
  39. Štrancar A, Perdith A (1992) Oximation of lignin carbonyl groups and their potential use for lignin determination. Holzforschung 46: 391–393CrossRefGoogle Scholar
  40. Takei T, Kato N, Iijima T, Higaki M (1995) Raman spectroscopic analysis of wood and bamboo lignin. Mokuzai Gakkaishi 41: 229–236Google Scholar
  41. Thomas BB (1970) Pulp properties. In: Britt KW (ed) Handbook of pulp and paper technology. Van Nostrand Reinhold Co., New York, pp 225–238Google Scholar
  42. Whetton R, Sederoff R (1991) Genetic engineering of wood. Forest Ecol Manage 43: 301–316CrossRefGoogle Scholar
  43. Williams HD, Fleming I (1976) Spectroscopic methods in organic chemistry. McGraw-Hill, Maidenhead, pp 35–73Google Scholar

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • Derek Stewart
    • 1
  • Nabila Yahiaoui
    • 2
  • Gordon J. McDougall
    • 1
  • Kate Myton
    • 2
  • Christiané Marque
    • 2
  • Alain M. Boudet
    • 2
  • James Haigh
    • 3
  1. 1.Unit for Industrial Crops, Department of Cellular and Environmental PhysiologyScottish Crop Research InstituteDundeeUK
  2. 2.Unité Mixte de Recherche UPS-CNRS No 5546University Paul SabatierToulouse CedexFrance
  3. 3.Department of ChemistryUniversity of SouthamptonSouthamptonUK

Personalised recommendations