, Volume 225, Issue 1, pp 23–39 | Cite as

Evidence for a role of AtCAD 1 in lignification of elongating stems of Arabidopsis thaliana

  • Aymerick Eudes
  • Brigitte Pollet
  • Richard Sibout
  • Cao-Trung Do
  • Armand Séguin
  • Catherine Lapierre
  • Lise JouaninEmail author
Original Article


The cinnamyl alcohol dehydrogenase (AtCAD) multigene family in Arabidopsis is composed of nine genes. Our previous studies focused on the two isoforms AtCAD C and AtCAD D which show a high homology to those related to lignification in other plants. This study focuses on the seven other Arabidopsis CAD for which functions are not yet elucidated. Their expression patterns were determined in different parts of Arabidopsis. Only CAD 1 protein can be detected in elongating stems, flowers, and siliques using Western-blot analysis. Tissue specific expression of CAD 1, B1, and G genes was determined using their promoters fused to the GUS reporter gene. CAD 1 expression was observed in primary xylem in accordance with a potential role in lignification. Arabidopsis T-DNA mutants knockout for the different genes CAD genes were characterized. Their stems displayed no substantial reduction of CAD activities for coniferyl and sinapyl alcohols as well as no modifications of lignin quantity and structure in mature inflorescence stems. Only a small reduction of lignin content could be observed in elongating stems of Atcad 1 mutant. These CAD genes in combination with the CAD D promoter were used to complement a CAD double mutant severely altered in lignification (cad c cad d). The expression of AtCAD A, B1, B2, F, and G had no effect on restoring a normal lignin profile of this mutant. In contrast, CAD 1 complemented partly this mutant as revealed by the partial restoration of conventional lignin units and by the decrease in the frequency of β-O-4 linked p-OH cinnamaldehydes.


Arabidopsis Cinnamyl alcohol dehydrogenase Gene family Lignin Stem 



Cinnamyl alcohol dehydrogenase







Authors are grateful to Frédéric Legée for Klason lignin analysis, to Laurent Cézar for thioacidolysis analysis, to Hervé Ferry and Bruno Letarnec for plant cultivation in the greenhouse. We are also grateful to Claire Halpin (School of Life Sciences, Dundee, UK) and Laigeng Li (Michigan Technological University, Raleigh, NC, USA) for providing antibodies, to Tsuyohi Nakagawa for the vector pGWB3, and to Bertrand Dubreucq for help in the use of the shp1/shp2 mutant. This work was partly supported by grant from GENOPLANTE (Af2001 009).

Supplementary material

425_2006_326_MOESM1_ESM.doc (67 kb)
Supplementary material


  1. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom N, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657PubMedCrossRefGoogle Scholar
  2. Altschul S, Gish W, Miller W, Myers W, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedCrossRefGoogle Scholar
  3. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  4. Baerson SR, Sanchez-Moreiras A, Pedrol-Bonjoch N, Schulz M, Kagan IA, Agarwal AK, Reigosa MJ, Duke SO (2005) Detoxification and transcriptome response in Arabidopsis seedlings exposed to the allelochemical benzoxazolin-2(3H)-one. J Biol Chem 280:21867–21881PubMedCrossRefGoogle Scholar
  5. 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 a cinnamyl alcohol dehydrogenase in poplar. Plant Physiol 112:1479–1490PubMedGoogle Scholar
  6. Baucher M, Bernard-Vailhe MA, Chabbert B, Besle JM, Opsomer C, Van Montagu M, Botterman J (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
  7. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266PubMedGoogle Scholar
  8. Blanco-Portales R, Medina-Escobar N, Lopez-Reyes JA, Villalba JM, Moyano E, Caballero JL, Munoz-Blanco J (2002) Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria × ananassa cv. Chandler). J Exp Bot 53:1723–1734PubMedCrossRefGoogle Scholar
  9. Bomati EK, Noel JP (2005) Structural and kinetic basis for substrate selectivity in sinapyl alcohol dehydrogenase. Plant Cell 17:1598–1611PubMedCrossRefGoogle Scholar
  10. Bouché N, Bouchez D (2001) Arabidopsis gene knockout: phenotypes wanted. Curr Opin Plant Biol 4:111–117PubMedCrossRefGoogle Scholar
  11. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–252PubMedCrossRefGoogle Scholar
  12. Brill EM, Abrahams S, Hayes CM, Jenkins CL, Watson JM (1999) Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.). Plant Mol Biol 41:279–291PubMedCrossRefGoogle Scholar
  13. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116Google Scholar
  14. Cheong YH, Chang H-S, Gupta R, Wang X, Zhu T, Luan S (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129:661–677PubMedCrossRefGoogle Scholar
  15. Costa MA, Collins RE, Anterola AM, Cochane FC, Davin LB, Lewis NG (2003) An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof. Phytochemistry 64:1097–1112PubMedCrossRefGoogle Scholar
  16. Damiani I, Morreel K, Danoun S, Goeminne G, Yahiaoui N, Marque C, Kopka J, Messens E, Goffner D, Boerjan W, Boudet A-M, Rochange S (2005) Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem. Plant Mol Biol 59:753–769PubMedCrossRefGoogle Scholar
  17. Dence C (1992) Lignin determination. In: Dence C, Lin S (eds) Methods in lignin biochemistry. Springer, Berlin Heidelberg New York, pp 33–61Google Scholar
  18. Estelle M, Somerville C (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol Gen Genet 206:200–206CrossRefGoogle Scholar
  19. Ferrandiz C, Pelaz S, Yanofsky MF (1999) Control of carpel and fruit development in Arabidopsis. Annu Rev Biochem 68:321–354PubMedCrossRefGoogle Scholar
  20. Goujon T, Sibout R, Eudes A, MacKay J, Jouanin L (2003a) Genes involved in the biosynthesis of lignin precursors in Arabidopsis thaliana. Plant Physiol Biochem 41:677–687CrossRefGoogle Scholar
  21. Goujon T, Ferret V, Mila I, Pollet B, Ruel K, Burlat V, Joseleau J-P, Barrière Y, Lapierre C, Jouanin L (2003b) Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability. Planta 217:218–228Google Scholar
  22. Griffiths DW, Robestson GW, Shepherd T, Ramsay G (1999) Epicuticular waxes and volatiles from faba bean (Vicia faba) flowers. Phytochemistry 52:607–612CrossRefGoogle Scholar
  23. Halpin C, Knight ME, Foxon GA, Campbell MM, Boudet AM, Boon JJ, Chabbert B, Tollier MT, Schuch W (1994) Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase. Plant J 6:339–350CrossRefGoogle Scholar
  24. Jefferson R, Kavanagh T, Bevan M (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  25. Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich IE, Dangl JL (1992) Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11:4677–4684PubMedGoogle Scholar
  26. Kim H, Ralph J, Lu F, Pilate G, Leplé J-C, Pollet B, Lapierre C (2002) Identification of the structure and origin of thioacidolysis marker compounds for cinnamyl alcohol dehydrogenase deficiency in angiosperms. J Biol Chem 277:47412–47419PubMedCrossRefGoogle Scholar
  27. Kim S-J, Kim M-R, Bedgar D, Moinuddin SGA, Cardenas CL, Davin LB, Kang CCK, Lewis NG (2004) Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc Natl Acad Sci USA 101:1455–1460PubMedCrossRefGoogle Scholar
  28. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  29. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  30. Lapierre C, Pollet B, Rolando R (1995) New insights into the molecular architecture of hardwood lignins by chemical degradation methods. Res Chem Intermed 21:397–412CrossRefGoogle Scholar
  31. Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret VV, 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 119:153–164PubMedCrossRefGoogle Scholar
  32. Lauter F-R (1996) Root-specific expression of the LeRse-1 gene in tomato is induced by exposure of the shoot to light. Mol Gen Genet 252:751–754PubMedGoogle Scholar
  33. Li L, Cheng XF, Leshkevich J, Umezawa T, Harding SA, Chiang VL (2001) The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell 13:1567–1586PubMedCrossRefGoogle Scholar
  34. Liljegren SL, Ditta GS, Eshed Y, Sadvidge B, Bowman JL, Yanofsky MF (2000) Shatterproof MADS-box genes control seed dispersal in Arabidopsis. Nature 404:766–770PubMedCrossRefGoogle Scholar
  35. Lurin C, Jouanin L (1995) RFLP of RT-PCR products: application to the expression of CHS multigene family in poplar. Mol Breed 1:411–417CrossRefGoogle Scholar
  36. Lynch D, Lidgett A, Mclnnes R, Huxley H, Jones E, Mahoney N, Spangenberg G (2002) Isolation and characterization of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.). J Plant Physiol 159:653–660CrossRefGoogle Scholar
  37. Marathe R, Guan Z, Anandalakshmi R, Zhao H, Dinesh-Kumar SP (2004) Study of Arabidopsis thaliana resistome in response to cucumber mosaic virus infection using whole genome microarray. Plant Mol Biol 55:501–520PubMedCrossRefGoogle Scholar
  38. Mitchell HJ, Hall JL, Barber MS (1994) Elicitor-induced cinnamyl alcohol dehydrogenase activity in lignifying wheat (Triticum aestivum L.) leaves. Plant Physiol 104:551–556PubMedGoogle Scholar
  39. Montesano M, Hyytiäinen H, Wettstein R, Tapio Palva E (2003) A novel potato defence-related alcohol: NADP+ oxidoreductase induced in response to Erwinia carotovora. Plant Mol Biol 52:1177–1189CrossRefGoogle Scholar
  40. Nakazono M, Qiu F, Borsuk LA, Schnable PS (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal or vascular tissues of maize. Plant Cell 15:583–596PubMedCrossRefGoogle Scholar
  41. Oh S, Park S, Han K-H (2003) Transcriptional regulation of secondary growth in Arabidopsis thaliana. J Exp Bot 5:2709–2722CrossRefGoogle Scholar
  42. Quirino BF, Normanly J, Amasino RM (1999) Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes. Plant Mol Biol 40:267–278PubMedCrossRefGoogle Scholar
  43. Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133:1051–1071PubMedCrossRefGoogle Scholar
  44. Rutledge RG, Côté C (2003) Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res 31:e93PubMedCrossRefGoogle Scholar
  45. Samson F, Brunaud V, Duchêne S, De Oliveira Y, Caboche M, Lecharny A, Aubourg S (2004) FLAGdb++: a database or the functional analysis of the Arabidopsis genome. Nucleic Acids Res 32(database issue):347–350CrossRefGoogle Scholar
  46. 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
  47. Sibout R, Eudes A, Mouille G, Pollet B, Lapierre C, Jouanin L, Séguin A (2005) Routing sinapyl and coniferyl alcohol pathway though the last reduction step in Arabidopsis thaliana using a double CAD mutant. Plant Cell 17:2059–2076PubMedCrossRefGoogle Scholar
  48. Somers DA, Nourse JP, Manners JM, Abrahms S, Watson JM (1995) A gene encoding a cinnamyl alcohol dehydrogenase homolog in Arabidopsis thaliana. Plant Physiol 108:1309–1310PubMedCrossRefGoogle Scholar
  49. Somssich IE, Bollman J, Hahlbrock K, Kombrink E, Schulz W (1989) Differential early activation of defense-related genes in elicitor-treated parsley cells. Plant Mol Biol 18:227–234CrossRefGoogle Scholar
  50. Somssich IE, Wernert P, Kiedrowski S, Hahlbrock K (1996) Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP(+) oxidoreductase. Proc Natl Acad Sci USA 93:14199–14203PubMedCrossRefGoogle Scholar
  51. Suh MC, Kim MJ, Hur C-C, Bae JM, Park YI, Chung C-H, Kang C-W, Ohlogge JB (2003) Comparative analysis of expressed sequence tags from Sesamun indicum and Arabidopsis thaliana developing seeds. Plant Mol Biol 52:1107–1123PubMedCrossRefGoogle Scholar
  52. 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
  53. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  54. Tobias M, Chow EK (2005) Structure of the cinnamyl alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification. Planta 220:678–688PubMedCrossRefGoogle Scholar
  55. Van Doorsselaere J, Baucher M, Feuillet C, Boudet AM, Van Montagu M, Inze D (1995) Isolation of cinnamyl alcohol dehydrogenase cDNAs from two important economic species: alfalfa and poplar. Demonstration of a high homology of the gene within angiosperms. Plant Physiol Biochem 33:105–109Google Scholar
  56. Verwoerd T, Dekker B, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362PubMedGoogle Scholar
  57. Vilaine F, Palauqui J-C, Amseleme J, Kusiak C, Lemoine R, Dinant S (2003) Towards deciphering phloem: a transcriptome analysis of the phloem of Apium graveolens. Plant J 36:67–81PubMedCrossRefGoogle Scholar
  58. Williamson JD, Stoop JM, Massel MO, Conkling MA, Pharr DM (1995) Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3. Proc Natl Acad Sci USA 92:7148–7152PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Aymerick Eudes
    • 1
  • Brigitte Pollet
    • 2
  • Richard Sibout
    • 3
    • 4
  • Cao-Trung Do
    • 1
  • Armand Séguin
    • 3
  • Catherine Lapierre
    • 2
  • Lise Jouanin
    • 1
    Email author
  1. 1.Biologie Cellulaire, INRAVersailles CedexFrance
  2. 2.Chimie Biologique, INRA-INA PGThiverval-GrignonFrance
  3. 3.Natural Resources Canada, Canadian Forest ServiceLaurentian Forestry CentreQCCanada
  4. 4.Biologie moléculaire végétaleBatiment Le BiophoreLausanneSwitzerland

Personalised recommendations