Plant Molecular Biology Reporter

, Volume 31, Issue 5, pp 1089–1099 | Cite as

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

  • Yi Xu
  • Shivegowda Thammannagowda
  • Tina P. Thomas
  • Parastoo Azadi
  • Scott E. Schlarbaum
  • Haiying LiangEmail author
Original Paper


Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.


Cinnamyl alcohol dehydrogenase (CAD) Lignin biosynthesis pyMBMS GUS reporter system 



The authors would like to thank Dr. Armand Séguina (Canadian Forest Service’s Laurentian Forestry Centre) and Dr. Richard Sibout (University of Lausanne, Switzerland) for providing the cad c cad d Arabidopsis double mutant plants and Dr. Chung-Jui Tsai at University of Georgia for her assistance with the PyMBMS analysis. This study was supported by a National Institute of Food and Agriculture/USDA grant (project number SC-1700324, technical contribution No. 6061 of the Clemson University Experiment Station) and an investment award provided by Clemson University.

Supplementary material

11105_2013_578_MOESM1_ESM.docx (1 mb)
ESM 1 (DOCX 1073 kb)


  1. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201PubMedCrossRefGoogle Scholar
  2. Baker AJ (1983) Wood fuel properties and fuel products from woods. In: Fuel wood management and utilization seminar. Michigan State University, East Lansing, pp 14–25Google Scholar
  3. Bateman RM, Crane PR, DiMichele WA, Kenrick PR, Rowe NP, Speck T, Stein WE (1998) Early evolution of land plants: phylogeny, physiology, and ecology of the primary terrestrial radiation. Ann Rev Ecol Syst 29:263–292CrossRefGoogle Scholar
  4. Baucher M, Chabbert B, Pilate G, van Doorsselaere J, Tollier MT, Petit-Conil M, Cornu D, Monties B, Van Montagu M, Inze D, Jouanin L, Boerjan W (1996) Red xylem and higher lignin extractability by down-regulating cinnamyl alcohol dehydrogenase in poplar (Populus tremula and Populus alba). Plant Physiol 112:1479–1490PubMedGoogle Scholar
  5. Baucher M, Halpin C, Petit-Conil M, Boerjan W (2003) Lignin: genetic engineering and impact on pulping. Crit Rev Biochem Mol Biol 38:305–350Google Scholar
  6. Berlin A, Gilkes N, Kilburn D, Maximenko V, Bura R, Markov A, Skomarovsky A, Okunev O, Gusakov A, Gregg D, Sinitsyn A, Saddler J (2006) Evaluation of cellulase preparations for hydrolysis of hardwood substrates. Appl Biochem Biotechnol 129:528–545PubMedCrossRefGoogle Scholar
  7. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546PubMedCrossRefGoogle Scholar
  8. Bomati EK, Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase. Plant Cell 17:1598–1611PubMedCrossRefGoogle Scholar
  9. Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510PubMedGoogle Scholar
  10. Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14PubMedCrossRefGoogle Scholar
  11. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17PubMedCrossRefGoogle Scholar
  12. Desfeux C, Clough SJ, Bent AF (2000) Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895–904PubMedCrossRefGoogle Scholar
  13. Eudes A, Pollet B, Sibout R, Do C-T, Séguin A, Lapierre C, Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana. Planta 225:23–39PubMedCrossRefGoogle Scholar
  14. Fornaléa S, Capelladesa M, Encinab A, Wangc K, Irara S, Lapierre C, Ruele K, Joseleaue JP, Berenguera J, Puigdomènecha P, Rigaua J, Caparrós-Ruiza D (2012) Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase. Mol Plant 5:817–830CrossRefGoogle Scholar
  15. Harlow WM, Harrar ES (1969) Textbook of dendrology. McGraw-Hill, New York, p 512Google Scholar
  16. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci 94:2122–2127PubMedCrossRefGoogle Scholar
  17. Hunt D (1998) Magnolias and their allies. International Dendrology Society & Magnolia Society, England, p 304Google Scholar
  18. Hwang SS, Lee SJ, Kim HK, Ka JO, Kim KJ, Song HG (2008) Biodegradation and saccharification of wood chips of Pinus strobus and Liriodendron tulipifera by white rot fungi. J Microbiol Biotechnol 18:1819–1826PubMedGoogle Scholar
  19. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5:387–405CrossRefGoogle Scholar
  20. Jiao J, Wickett N, Ayyampalayam S, Chanderbali A, Landherr L, Ralph PE, Tomsho LP, Liang H, Soltis PS, Soltis DE, Clifton SE, Schlarblum SE, Shuster SC, Ma H, Leebens-Mack J, dePamphilis CW (2011) Phylogenomic detection of ancient polyploidy in seed plants and angiosperms. Nature 473:97–100PubMedCrossRefGoogle Scholar
  21. Jin J, Do J, Moon D, Noh EW, Kim W, Kwon M (2011) EST analysis of functional genes associated with cell wall biosynthesis and modification in the secondary xylem of the yellow poplar (Liriodendron tulipifera) stem during early stage of tension wood formation. Planta 234:959–977PubMedCrossRefGoogle Scholar
  22. Johnson TG, Bentley JW, Howell M (2011) The South’s timber industry—an assessment of timber product output and use, 2009. Department of Agriculture Forest Service, Southern Research Station, Asheville, p 44, Resour Bull SRS–182Google Scholar
  23. Kajita S, Katayama Y, Omori S (1996) Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate:coenzyme A ligase. Plant Cell Physiol 37:957–965PubMedCrossRefGoogle Scholar
  24. Kim KD, Lee EJ (2005) Potential tree species for use in the restoration of unsanitary landfills. Environ Manag 36:1–14CrossRefGoogle Scholar
  25. Kim SJ, Kim MR, Bedgar DL, Moinuddin SGA, Cardenas CL, Davin LB, Kang C, Lewis NF (2004) Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc Natl Acad Sci 101:1455–1460PubMedCrossRefGoogle Scholar
  26. Kim SJ, Kim KW, Cho MH, Franceschi VR, Davin LB, Lewis NG (2007) Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations? Phytochem 68:1957–1974CrossRefGoogle Scholar
  27. Lafayette PR, Eriksson KE, Dean JF (1999) Characterization and heterologous expression of laccase cDNAs from the lignifying xylem of yellow-poplar (Liriodendron tulipifera). Plant Mol Biol 40:23–35PubMedCrossRefGoogle Scholar
  28. Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret V, De Nadai V, Jouanin L (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
  29. Larionov A, Krause A, Miller W (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinforma 6:62CrossRefGoogle Scholar
  30. Li X, Weng J-K, Chapple C (2008) Improvement of biomass through lignin modification. Plant J 54:569–581PubMedCrossRefGoogle Scholar
  31. Liang H, Ayyampalayam S, Wickett N, Barakat A, Xu Y, Landherr L, Ralph P, Xu T, Schlarbaum SE, Leebens-Mack JH, dePamphilis CW (2011) Generation of a large-scale genomic resource for functional and comparative genomics in Liriodendron. Tree Genet Genomes 7:941–954CrossRefGoogle Scholar
  32. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M (2009) Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels. New Phytol 182:878–890PubMedCrossRefGoogle Scholar
  33. Persson B, Zigler JS, Jörnvall H (1994) A super-family of medium-chain dehydrogenases/reductases (MDR). Sub-lines including ζ-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductases, enoyl reductases, VAT-1 and other proteins. Eur J Biochem 226:15–22PubMedCrossRefGoogle Scholar
  34. Pilate G, Guieny E, Holt K, Petit-Conil M, Lapierre C, Leple JC, Pollet B, Mila I, Webster EA, Marstorp G et al (2002) Field and pulping performances of transgenic trees with altered lignification. Nat Biotechnol 20:607–612PubMedCrossRefGoogle Scholar
  35. Rao ST, Rossmann MG (1973) Comparison of super-secondary structures in proteins. J Mol Biol 76:241–256PubMedCrossRefGoogle Scholar
  36. Ronse de Craene L, Soltis DE, Soltis PS (2003) Evolution of floral structures in basal angiosperms. Int J Plant Sci 164:S329–S363CrossRefGoogle Scholar
  37. 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 181:783–795PubMedCrossRefGoogle Scholar
  38. Sibout R, Eudes A, Pollet B, Goujon T, Mila I, Granier F, Séguin A, Lapierre C, Jouanin L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis: isolation and characterization of the corresponding mutants. Plant Physiol 132:848–860PubMedCrossRefGoogle Scholar
  39. Sibout R, Eudes A, Mouilleb G, Polletc B, Lapierre C, Jouaninb L, Séguin A (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
  40. Solovyev VV, Shahmuradov IA (2003) PromH: promoters identification using orthologous genomic sequences. Nucleic Acids Res 31:3540–3545PubMedCrossRefGoogle Scholar
  41. Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102–1104PubMedGoogle Scholar
  42. Sykes R, Kodrzycki B, Tuskan G, Foutz K, Davis M (2008) Within tree variability of lignin composition in Populus. Wood Sci Technol 42:649–661CrossRefGoogle Scholar
  43. Tavares R, Aubourg S, Lecharny A, Kreis M (2000) Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2. Plant Mol Biol 42:703–717PubMedCrossRefGoogle Scholar
  44. Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  45. Vanholme R, Demedts B, Morreel K, Ralph J, Boerja W (2010) Lignin biosynthesis and structure. Plant Physiol 153:895–905PubMedCrossRefGoogle Scholar
  46. Weng JK, Chapple C (2010) The origin and evolution of lignin biosynthesis. New Phytol 187:273–285PubMedCrossRefGoogle Scholar
  47. Williams RS, Feist WC (2004) Durability of yellow-poplar and sweetgum and service life of finishes after long-term exposure. For Prod J 54:96–101Google Scholar
  48. Xu Z, Zhang D, Hu J, Zhou X, Ye X, Reichel KL, Stewart NR, Syrenne RD, Yang X, Gao P, Shi W, Doeppke C, Sykes RW, Burris JN, Bozell JJ, Cheng ZM, Hayes DG, Labbe N, Davis M, Stewart CN, Yuan JS (2009) Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom. BMC Bioinforma 10(Suppl 11):S3CrossRefGoogle Scholar
  49. Xu Y, Schlarbaum SE, Liang H (2011) Investigation of genome structure of a cinnamyl alcohol dehydrogenase locus in a basal angiosperm hardwood species, Liriodendron tulipifera L., reveals low synteny. J Syst Evol 9:396–405CrossRefGoogle Scholar
  50. Youn B, Camacho R, Moinuddin SGA, Lee C, Davin LB, Lewis NG, Kang C (2006) Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4. Org Biomol Chem 4:1687–1697PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Yi Xu
    • 1
  • Shivegowda Thammannagowda
    • 1
  • Tina P. Thomas
    • 2
  • Parastoo Azadi
    • 3
  • Scott E. Schlarbaum
    • 4
  • Haiying Liang
    • 1
    Email author
  1. 1.Department of Genetics and BiochemistryClemson UniversityClemsonUSA
  2. 2.Bioenergy Systems Research InstituteUniversity of GeorgiaAthensUSA
  3. 3.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  4. 4.Department of Forestry, Wildlife & FisheriesThe University of TennesseeKnoxvilleUSA

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