Journal of Plant Research

, 119:581 | Cite as

Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells

Regular Paper


We previously showed that eight laccase genes (Lac 1Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147–155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO4 in the reaction mixtures increased in the range from 1 to 100 μM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.


Bright yellow-2 (BY-2) Dilignols Heterologous expression Lignin synthesis Multicopper oxidase 



We are grateful to Dr. Yuko Ohashi of the National Institute of Agrobiological Sciences for providing a binary vector pBE2113-GUS, Dr. Tsuyoshi Kaneta of Ehime University for technical help in transformation of tobacco BY-2 cells, and Professor Masahiro Inouhe of Ehime University for use of equipment. This work was supported in part by a project grant from Ehime University (Rudimentary Research Support to Y.S.).


  1. Bailey MR, Woodard SL, Callaway E, Beifuss K, Magallanes-Lundback M, Lane JR, Horn ME, Mallubhotla H, Delaney DD, Ward M, Van Gastel F, Howard JA, Hood EE (2004) Improved recovery of active recombinant laccase from maize seed. Appl Microbiol Biotechnol 63:390–397PubMedCrossRefGoogle Scholar
  2. Baker SS, Rugh CL, Kamalay JC (1990) RNA and DNA isolation from recalcitrant plant tissues. BioTechniques 9:268–272PubMedGoogle Scholar
  3. Bao W, O’Malley DM, Whetten R, Sederoff RR (1993) A laccase associated with lignification in loblolly pine xylem. Science 260:636–638CrossRefGoogle Scholar
  4. Bligny R, Gaillard J, Douce R (1986) Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Effects of a copper deficiency. Biochem J 237:583–588PubMedGoogle Scholar
  5. Davin LB, Bedgar DL, Katayama T, Lewis NG (1992) On the stereospecific synthesis of (+) pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol. Phytochemistry 31:3869–3874PubMedCrossRefGoogle Scholar
  6. Demura T, Tashiro G, Horiguchi G, Kishimoto N, Kubo M, Matsuoka N, Minami A, Nagata-Hiwatashi M, Nakamura K, Okamura Y, Sassa N, Suzuki S, Yazaki J, Kikuchi S, Fukuda H (2002) Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells. Proc Natl Acad Sci USA 99:15794–15799PubMedCrossRefGoogle Scholar
  7. Driouich A, Laine AC, Vian B, Faye L (1992) Characterization and localization of laccase forms in stem and cell cultures of sycamore. Plant J 2:13–24CrossRefGoogle Scholar
  8. Friedrichsen DM, Nemhauser J, Muramitsu T, Maloof JN, Alonso J, Ecker JR, Furuya M, Chory J (2002) Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth. Genetics 162:1445–1456PubMedGoogle Scholar
  9. Fromard L, Babin V, Fleurat-Lessard P, Fromont J-C, Serrano R, Bonnemain J-L (1995) Control of vascular sap pH by the vessel-associated cells in woody species. Plant Physiol 108:913–918PubMedGoogle Scholar
  10. Lafayette PR, Eriksson K-EL, Dean JFD (1999) Characterization and heterologous expression of laccase cDNAs from xylem tissues yellow poplar (Liriodendron tulipifera). Plant Mol Biol 40:23–35PubMedCrossRefGoogle Scholar
  11. Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF (2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404:766–770PubMedCrossRefGoogle Scholar
  12. Mayer AM (1987) Polyphenol oxidases in plants—recent progress. Phytochemistry 26:11–20CrossRefGoogle Scholar
  13. McCaig BC, Meagher RB, Dean JF (2005) Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana. Planta 221:619–636PubMedCrossRefGoogle Scholar
  14. McDougall GJ, Morrison IM (1996) Extraction and partial purification of cell wall-associated coniferyl alcohol oxidase from developing xylem of Sitka spruce. Holzforschung 50:549–553CrossRefGoogle Scholar
  15. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedGoogle Scholar
  16. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Inter Rev Cytol 132:1–30CrossRefGoogle Scholar
  17. Nakamura K, Go N (2005) Function and molecular evolution of multicopper blue proteins. Cell Mol Life Sci 62:2050–2066PubMedCrossRefGoogle Scholar
  18. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405:200–203PubMedCrossRefGoogle Scholar
  19. Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet AM, Goffner D (1999) Biochemical characterization, molecular cloning and expression of laccases, a divergent gene family in poplar. Eur J Biochem 259:485–495PubMedCrossRefGoogle Scholar
  20. Ranocha P, Chabannes M, Chamayou S, Danoun S, Jauneau A, Boudet AM, Goffner D (2002) Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol 129:145–155PubMedCrossRefGoogle Scholar
  21. Sato Y, Sugiyama M, Górecki RJ, Fukuda H, Komamine A (1993) Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia. Planta 189:584–589CrossRefGoogle Scholar
  22. Sato Y, Sugiyama M, Komamine A, Fukuda H (1995) Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation. Planta 196:141–147CrossRefGoogle Scholar
  23. Sato Y, Wuli B, Sederoff R, Whetten R (2001) Molecular cloning and expression of eight laccase cDNAs in loblolly pine (Pinus taeda). J Plant Res 114:147–155CrossRefGoogle Scholar
  24. Sato Y, Demura T, Yamawaki K, Inoue Y, Sato S, Sugiyama M, Fukuda H (2006) Isolation and characterization of a novel peroxidase gene ZPO-C of which expression and function are closely associated with lignification during tracheary element differentiation. Plant Cell Physiol 47:493–503PubMedCrossRefGoogle Scholar
  25. Schell J (1997) Interdependence of pH, malate concentration, and calcium and magnesium concentrations in the xylem sap of beech roots. Tree Physiol 17:479–483PubMedGoogle Scholar
  26. Sterjiades R, Dean JFD, Eriksson K-EL (1992) Laccase from sycamore maple (Acer pseudoplatanus) polymerizes monolignols. Plant Physiol 99:1162–1168PubMedCrossRefGoogle Scholar
  27. Sterjiades R, Ranocha P, Boudet AM, Goffner D (1996) Identification of specific laccases isoforms capable of polymerizing monolignols by an “in gel” procedure. Anal Biochem 242:158–161PubMedCrossRefGoogle Scholar
  28. Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26CrossRefGoogle Scholar
  29. Tokunaga N, Sakakibara N, Umezawa T, Ito Y, Fukuda H, Sato Y (2005) Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells. Plant Cell Physiol 46:224–232PubMedCrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of Biology, Graduate School of Science and EngineeringEhime UniversityMatsuyamaJapan
  2. 2.Department of Forestry and Environmental ResourcesNorth Carolina State UniversityRaleighUSA

Personalised recommendations