BioEnergy Research

, 1:180 | Cite as

Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

  • Lisa A. Jackson
  • Gail L. Shadle
  • Rui Zhou
  • Jin Nakashima
  • Fang Chen
  • Richard A. Dixon


A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.


Alfalfa Genetic modification Lignin pathway Saccharification Transgenic plants 



cinnamyl alcohol dehydrogenase


cinnamoyl coenzyme A reductase




hydroxycinnamoyl transferase



Supplementary material

12155_2008_9020_MOESM1_ESM.doc (410 kb)
ESM 1Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. (DOC 410 KB)


  1. 1.
    Baucher M, BernardVailhe MA, Chabbert B et al (1999) Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol 39:437–447PubMedCrossRefGoogle Scholar
  2. 2.
    Baucher M, Chabbert B, Pilate G et al (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar. Plant Physiol 112:1479–1490PubMedGoogle Scholar
  3. 3.
    Bernard-Vailhé MA, Besle JM, Maillot MP et al (1998) Effect of down-regulation of cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems. J Sci Food Agric 76:505–514CrossRefGoogle Scholar
  4. 4.
    Bessau S, Hoffmann L, Geoffroy P et al (2007) Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19:148–162CrossRefGoogle Scholar
  5. 5.
    Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546PubMedCrossRefGoogle Scholar
  6. 6.
    Boudet AM, Kajita S, Grima-Pettenati J et al (2003) Lignins and lignocellulosics: a better control of synthesis for new and improved uses. Trends Plant Sci 8:576–581PubMedCrossRefGoogle Scholar
  7. 7.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. 8.
    Chabannes M, Barakate A, Lapierre C et al (2001a) Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J 28:257–270PubMedCrossRefGoogle Scholar
  9. 9.
    Chabannes M, Ruel K, Yoshinaga A et al (2001b) In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels. Plant J 28:271–282PubMedCrossRefGoogle Scholar
  10. 10.
    Chen F, Dixon RA (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotech 25:759–761CrossRefGoogle Scholar
  11. 11.
    Chen F, Reddy MSS, Temple S et al (2006) Multi-site genetic modulation of monolignol biosynthesis suggests new routes for the formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.). Plant J 48:113–124PubMedCrossRefGoogle Scholar
  12. 12.
    Chen L, Auh C-K, Dowling P et al (2003) Improved forgage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnol J 1:437–449PubMedCrossRefGoogle Scholar
  13. 13.
    Davison BH, Drescher SR, Tuskan GA et al (2006) Variation of S/G ratio and lignin content in a Populus family influences the release of fermentable sugars by dilute acid hydrolysis. Appl Biochem Biotechnol 129–132:427–435PubMedCrossRefGoogle Scholar
  14. 14.
    Dien BS, Jung H-JG, Vogel KP et al (2006) Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass Bioenergy 30:880–891CrossRefGoogle Scholar
  15. 15.
    Do C-T, Pollet B, Thevenin J et al (2007) Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid 3-O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 226:1117–1129PubMedCrossRefGoogle Scholar
  16. 16.
    Dubois M, Gilles KA, Hamilton JK et al (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  17. 17.
    Ebert J (2007) Alfalfa’s bioenergy appeal. Ethanol Producer Magazine (BBI International), Sept 2007, pp 88–94Google Scholar
  18. 18.
    Eudes A, Pollet B, Sibout R et al (2006) Evidence for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana. Planta 225:23–39PubMedCrossRefGoogle Scholar
  19. 19.
    Goujon T, Ferret V, Mila I et al (2003) Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effect on phenotype, lignins and cell wall degradability. Planta 217:218–228PubMedGoogle Scholar
  20. 20.
    Guo D, Chen F, Inoue K et al (2001a) Down-regulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa (Medicago sativa L.): impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell 13:73–88PubMedCrossRefGoogle Scholar
  21. 21.
    Guo D, Chen F, Wheeler J et al (2001b) Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res 10:457–464PubMedCrossRefGoogle Scholar
  22. 22.
    Halpin C, Knight ME, Foxon GA et al (1994) Manipulation of lignin quality by down-regulation of cinnamyl alcohol dehydrogenase. Plant J 6:339–350CrossRefGoogle Scholar
  23. 23.
    Hein JJ (1990) Unified approach to alignment and phylogenies. Meth Enzymol 183:626–645PubMedCrossRefGoogle Scholar
  24. 24.
    Hibino T, Yakabe K, Kawazu T et al (1995) Increase of cinnamaldehyde groups in lignin of transgenic tobacco plants carrying an antisense gene for cinnamyl alcohol dehydrogenase. Biosci Biotech Biochem 59:929–931Google Scholar
  25. 25.
    Hoffmann L, Maury S, Martz F et al (2003) Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem 278:95–103PubMedCrossRefGoogle Scholar
  26. 26.
    Humphreys JM, Chapple C (2002) Rewriting the lignin roadmap. Curr Opin Plant Biol 5:224–229PubMedCrossRefGoogle Scholar
  27. 27.
    Jones L, Ennos AR, Turner SR (2001) Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J 26:205–216PubMedCrossRefGoogle Scholar
  28. 28.
    Lamb JFS, Sheaffer CC, Samac DA (2003) Population density and harvest maturity effects on leaf and stem yield in alfalfa. Agron J 95:635–641Google Scholar
  29. 29.
    Lapierre C, Monties B, Rolando C (1985) Thioacidolysis of lignin: comparison with acidolysis. J Wood Chem Technol 5:277–292CrossRefGoogle Scholar
  30. 30.
    Lapierre C, Pilate G, Pollet B et al (2004) Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins. Phytochemistry 65:313–321PubMedCrossRefGoogle Scholar
  31. 31.
    Lapierre C, Pollet B, MacKay JJ et al (2000) Lignin structure in a mutant pine deficient in cinnamyl alcohol dehydrogenase. J Agric Food Chem 48:2326–2331PubMedCrossRefGoogle Scholar
  32. 32.
    Lapierre C, Pollet B, PetitConil M et al (1999) Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol 119:153–163PubMedCrossRefGoogle Scholar
  33. 33.
    Lapierre C, Pollet B, Rolando C (1995) New insight into the molecular architecture of hardwood lignins by chemical degradative method. Res Chem Intermed 21:397–412CrossRefGoogle Scholar
  34. 34.
    Leple JC, Dauwe R, Morreel K et al (2007) Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell 19:3669–3691PubMedCrossRefGoogle Scholar
  35. 35.
    Luo C, Brink DL, Blanch HW (2002) Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol. Biomass Bioenergy 22:125–138CrossRefGoogle Scholar
  36. 36.
    Nakashima J, Chen F, Jackson L et al (2008) Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa L.): effects on lignin composition in specific cell types. New Phytol 179:738–750PubMedCrossRefGoogle Scholar
  37. 37.
    O’Connell A, Holt K, Piquemal J et al (2002) Improved paper pulp from plants with suppressed cinnamoyl-CoA reductase or cinnamyl alcohol dehydrogenase. Transgenic Res 11:495–503PubMedCrossRefGoogle Scholar
  38. 38.
    Oldroyd GE, Geurts R (2001) Medicago truncatula, going where no plant has gone before. Trends Plant Sci 6:552–554PubMedCrossRefGoogle Scholar
  39. 39.
    Parvathi K, Chen F, Guo D et al (2001) Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant J 25:193–202PubMedCrossRefGoogle Scholar
  40. 40.
    Pilate G, Guiney E, Holt K et al (2002) Field and pulping performances of transgenic trees with altered lignification. Nat Biotechnol 20:607–612PubMedCrossRefGoogle Scholar
  41. 41.
    Piquemal J, Lapierre C, Myton K (1998) Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant J 13:71–83CrossRefGoogle Scholar
  42. 42.
    Ragauskas AJ, Williams CK, Davison BH et al (2006) The path forward for biofuels and biomaterials. Science 311:484–489PubMedCrossRefGoogle Scholar
  43. 43.
    Ralph J, Hatfield RD, Piquemal J et al (1998) NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamyl-alcohol dehydrogenase and cinnamoyl-CoA reductase. Proc Natl Acad Sci USA 95:12803–12808PubMedCrossRefGoogle Scholar
  44. 44.
    Reddy MSS, Chen F, Shadle GL et al (2005) Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proc Natl Acad Sci USA 102:16573–16578PubMedCrossRefGoogle Scholar
  45. 45.
    Reddy MSS, Ghabrial SA, Redmond CT et al (2002) Resistance to bean pod mottle virus in transgenic soybean lines expressing the capsid polyprotein. Phytopathology 91:831–838CrossRefGoogle Scholar
  46. 46.
    Rolando C, Monties B, Lapierre C (1992) Thioacidolysis. In: Lin SY, Dence CW (eds) Methods in lignin chemistry. Springer-Verlag, Berlin Heidelberg, pp 334–340Google Scholar
  47. 47.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  48. 48.
    Shadle G, Chen F, Reddy MSS et al (2006) Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxy cinnamoyl transferase in transgenic alfalfa impacts lignification, development and forage quality. Phytochemistry 68:1521–1529CrossRefGoogle Scholar
  49. 49.
    Sibout R, Eudes A, Mouille G et al (2005) Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17:2059–2076PubMedCrossRefGoogle Scholar
  50. 50.
    van der Rest B, Danoun S, Boudet AM et al (2006) Down-regulation of cinnamoyl-CoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes in soluble phenolic pools. J Exp Bot 57:1399–1411PubMedCrossRefGoogle Scholar
  51. 51.
    Vogel KP, Jung HJG (2001) Genetic modification of herbaceous plants for feed and fuel. Crit Rev Plant Sci 20:15–49CrossRefGoogle Scholar
  52. 52.
    Vogel KP, Pederson JF, Masterson SD, Toy JJ (1999) Evaluation of a filterbag system for NDF, ADF, and IVDMD forage analysis. Crop Sci 39:276–279Google Scholar
  53. 53.
    Wyrambik D, Grisebach H (1979) Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Eur J Biochem 97:503–509PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Lisa A. Jackson
    • 1
  • Gail L. Shadle
    • 1
  • Rui Zhou
    • 1
  • Jin Nakashima
    • 1
  • Fang Chen
    • 1
  • Richard A. Dixon
    • 1
  1. 1.Plant Biology Division, Samuel Roberts Noble FoundationArdmoreUSA

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