Skip to main content
SpringerLink
Log in

Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

BioEnergy Research Aims and scope Submit manuscript

Abstract

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price includes VAT (France)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Keshwani DR, Cheng JJ (2009) Switchgrass for bioethanol and other value-added applications: a review. Bioresour Technol 100:1515–1523

    Article  PubMed  CAS  Google Scholar 

  2. McLaughlin SB, Kszos LA (2005) Development of switchgrass (Panicum virgatum) as a bioenergy feedstock in the United States. Biomass Bioenergy 28:515–535

    Article  Google Scholar 

  3. Bouton JH (2007) Molecular breeding of switchgrass for use as a biofuel crop. Curr Opin Genet Dev 17:553–558

    Article  PubMed  CAS  Google Scholar 

  4. Schmer MR, Vogel KP, Mitchell RB, Perrin RK (2008) Net energy of cellulosic ethanol from switchgrass. Proc Natl Acad Sci USA 105:464–469

    Article  PubMed  CAS  Google Scholar 

  5. Dien BS, Jung H-JG, Vogel KP, Casler MD, Lamb JFS, Iten L et al (2006) Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass Bioenergy 30:880–891

    Article  CAS  Google Scholar 

  6. Chen F, Dixon RA (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol 25:759–761

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  8. Sticklen MB (2008) Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 9:433–443

    Article  PubMed  CAS  Google Scholar 

  9. Ralph J, Akiyama T, Kim H, Lu F, Schatz PF, Marita JM et al (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281:8843–8853

    Article  PubMed  CAS  Google Scholar 

  10. Hisano H, Nandakumar R, Wang Z-Y (2009) Genetic modification of lignin biosynthesis for improved biofuel production. In Vitro Cell Dev Biol Plant 45:306–313

    Article  CAS  Google Scholar 

  11. Rogers LA, Campbell MMC (2004) The genetic control of lignin deposition during plant growth and development. New Phytol 164:30

    Article  Google Scholar 

  12. Baucher M, Monties B, Van Montagu M, Boerjan W (1998) Biosynthesis and genetic engineering of lignin. Crit Rev Plant Sci 17:125–197

    Article  CAS  Google Scholar 

  13. Bernard-Vailhe MA, Besle JM, Maillot MP, Cornu A, Halpin C, Knight M (1998) Effect of down-regulation of cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems. J Sci Food Agric 76:505–514

    Article  Google Scholar 

  14. Yahiaoui N, Marque C, Myton KE, Negrel J, Boudet AM (1998) Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants. Planta 204:8–15

    Article  CAS  Google Scholar 

  15. Baucher M, Bernard Vailhe MA, Chabbert B, Besle JM, Opsomer C, Van Montagu M 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 39:437–447

    Article  PubMed  CAS  Google Scholar 

  16. Jackson L, Shadle G, Zhou R, Nakashima J, Chen F, Dixon R (2008) Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. Bioenerg Res 1:180–192

    Article  Google Scholar 

  17. Baucher M, Chabbert B, Pilate G, Tollier MT, Petit Conil M, Cornu D et al (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar. Plant Physiol 112:1479–1490

    PubMed  CAS  Google Scholar 

  18. Lapierre C, Pollet B, Petit Conil M, Toval G, Romero J, Pilate G 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 119:153–163

    Article  PubMed  CAS  Google Scholar 

  19. Pilate G, Guiney E, Holt K, PetitConil M, Lapierre C, Leple JC et al (2002) Field and pulping performances of transgenic trees with altered lignification. Nat Biotechnol 20:607–612

    Article  PubMed  CAS  Google Scholar 

  20. Valério L, Carter D, Rodrigues JC, Tournier V, Gominho J, Marque C et al (2003) Down regulation of cinnamyl alcohol dehydrogenase, a lignification enzyme, in Eucalyptus camaldulensis. Mol Breed 12:157–167

    Article  Google Scholar 

  21. Wróbel-Kwiatkowska M, Starzycki M, Zebrowski J, Oszmianski J, Szopa J (2007) Lignin deficiency in transgenic flax resulted in plants with improved mechanical properties. J Biotechnol 128:919–934

    Article  PubMed  Google Scholar 

  22. Chen L, Auh C, Dowling P, Bell J, Chen F, Hopkins A et al (2003) Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnol J 1:437–449

    Article  PubMed  CAS  Google Scholar 

  23. Sattler SE, Saathoff AJ, Haas EJ, Palmer NA, Funnell-Harris DL, Sarath G et al (2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is responsible for the sorghum brown midrib6 phenotype. Plant Physiol 150:584–595

    Article  PubMed  CAS  Google Scholar 

  24. Saballos A, Ejeta G, Sanchez E, Kang C, Vermerris W (2009) A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib6 gene. Genetics 181:783–795

    Article  PubMed  CAS  Google Scholar 

  25. Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B et al (1998) Brown-midrib maize (bm1) - a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J 14:545–553

    Article  PubMed  CAS  Google Scholar 

  26. Wang Z-Y, Ge Y (2006) Recent advances in genetic transformation of forage and turf grasses. In Vitro Cell Dev Biol Plant 42:1–18

    Google Scholar 

  27. Moore KJ, Moser LE, Vogel KP, Waller SS, Johnson BE, Pedersen JF (1991) Describing and quantifying growth stages of perennial forage grasses. Agron J 83:1073–1077

    Article  Google Scholar 

  28. Kumar S, Tamura K, Nei M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

    Article  PubMed  CAS  Google Scholar 

  29. Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495

    Article  PubMed  CAS  Google Scholar 

  30. Xi Y, Fu C, Ge Y, Nandakumar R, Hisano H, Bouton J et al (2009) Agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes. Bioenerg Res 2:275–283

    Article  Google Scholar 

  31. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66

    Article  PubMed  CAS  Google Scholar 

  32. Ranasinghe S, Rogers ME, Hamilton JGC, Bates PA, Maingon RDC (2008) A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis. Trans R Soc Trop Med Hyg 102:875–882

    Article  PubMed  CAS  Google Scholar 

  33. Zhang K, Qian Q, Huang Z, Wang Y, Li M, Hong L et al (2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice. Plant Physiol 140:972–983

    Article  PubMed  CAS  Google Scholar 

  34. 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 72:248–254

    Article  PubMed  CAS  Google Scholar 

  35. dos Santos WD, MdLL F, Ferrarese-Filho O (2006) High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots. Plant Physiol Biochem 44:511–515

    Article  PubMed  Google Scholar 

  36. Chen L, Auh C, Chen F, Cheng XF, Aljoe H, Dixon RA et al (2002) Lignin deposition and associated changes in anatomy, enzyme activity, gene expression and ruminal degradability in stems of tall fescue at different developmental stages. J Agric Food Chem 50:5558–5565

    Article  PubMed  CAS  Google Scholar 

  37. Hatfield RD, Grabber J, Ralph J, Brei K (1999) Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. J Agric Food Chem 47:628–632

    Article  PubMed  CAS  Google Scholar 

  38. Lapierre C, Pollet B, Rolando C (1995) New insight into the molecular architecture of hardwood lignins by chemical degradative method. Res Chem Intermed 21:397–412

    Article  CAS  Google Scholar 

  39. Sarath G, Baird LM, Vogel KP, Mitchell RB (2007) Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum L.). Bioresour Technol 98:2985–2992

    Article  PubMed  CAS  Google Scholar 

  40. Shen H, Fu C, Xiao X, Ray T, Tang Y, Wang Z et al (2009) Developmental control of lignification in stems of lowland switchgrass variety Alamo and the effects on saccharification efficiency. Bioenerg Res 2:233–245

    Article  Google Scholar 

  41. Huhman DV, Berhow MA, Sumner LW (2005) Quantification of saponins in aerial and subterranean tissues of Medicago truncatula. J Agric Food Chem 53:1914–1920

    Article  PubMed  CAS  Google Scholar 

  42. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268

    Article  CAS  Google Scholar 

  43. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    Article  CAS  Google Scholar 

  44. Kim S-J, Kim M-R, Bedgar DL, Moinuddin SGA, Cardenas CL, Davin LB et al (2004) Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc Natl Acad Sci USA 101:1455–1460

    Article  PubMed  CAS  Google Scholar 

  45. Saathoff AJ, Tobias CM, Sattler SE, Haas EJ, Twigg P, Sarath G (2010) Switchgrass contains two cinnamyl alcohol dehydrogenases involved in lignin formation. Bioenerg Res. doi:10.1007/s12155-010-9106-2

    Google Scholar 

  46. Bernard-Vailhe MA, Cornu A, Robert D, Maillot MP, Besle JM (1996) Cell wall degradability of transgenic tobacco stems in relation to their chemical extraction and lignin quality. J Agric Food Chem 44:1164–1169

    Article  CAS  Google Scholar 

  47. Palmer N, Sattler S, Saathoff A, Funnell D, Pedersen J, Sarath G (2008) Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum. Planta 229:115–127

    Article  PubMed  CAS  Google Scholar 

  48. Reddy MSS, Chen F, Shadle G, Jackson L, Aljoe H, Dixon RA (2005) Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proc Natl Acad Sci USA 102:16573–16578

    Article  PubMed  CAS  Google Scholar 

  49. Niggeweg R, Michael AJ, Martin C (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol 22:746–754

    Article  PubMed  CAS  Google Scholar 

  50. Boudet AM, Kajita S, Grima-Pettenati J, Goffner D (2003) Lignins and lignocellulosics: a better control of synthesis for new and improved uses. Trends Plant Sci 8:576–581

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Ko Shimamoto for providing the pANDA vector, Human David for assistance with GC-MS and LC-MS analysis, Stacy Allen and Tui Ray for assistance with real-time RT-PCR analysis, and Dennis Walker for assistance with forage digestibility analysis. The work was supported by the US Department of Agriculture and US Department of Energy Biomass Initiative (project no. 2009-10003-05140), the BioEnergy Science Center and the Samuel Roberts Noble Foundation. The BioEnergy Science Center is supported by the Office of Biological and Environmental Research in the DOE Office of Science.

Author information

Authors and Affiliations

  1. Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    Chunxiang Fu, Xirong Xiao, Yajun Xi, Yaxin Ge, Joseph Bouton & Zeng-Yu Wang

  2. Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    Fang Chen & Richard A. Dixon

  3. BioEnergy Science Center, Oak Ridge, TN, 37831, USA

    Xirong Xiao, Fang Chen, Richard A. Dixon & Zeng-Yu Wang

Authors
  1. Chunxiang Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xirong Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yajun Xi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yaxin Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joseph Bouton
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Richard A. Dixon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zeng-Yu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zeng-Yu Wang.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Table S1

Content of cell wall-bound phenolic compounds in internodes of transgenic switchgrass. (DOCX 12 kb)

Table S2

Cell wall polysaccharides and in vitro true dry matter digestibility (IVTDMD) of transgenic switchgrass plants with downregulated expression of CAD. (DOCX 12 kb)

Supplementary Figure

(PPTX 146 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fu, C., Xiao, X., Xi, Y. et al. Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass. Bioenerg. Res. 4, 153–164 (2011). https://doi.org/10.1007/s12155-010-9109-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12155-010-9109-z

Keywords

search

Navigation