Journal of Wood Science

, Volume 56, Issue 6, pp 460–469 | Cite as

Overexpression of a fungal laccase gene induces nondehiscent anthers and morphological changes in flowers of transgenic tobacco

  • Zannatul Nasrin
  • Misato Yoshikawa
  • Yuki Nakamura
  • Shahanara Begum
  • Satoshi Nakaba
  • Mikiko Uesugi
  • Yuriko Osakabe
  • Tomonori Sonoki
  • Kanna Sato
  • Ryo Funada
  • Yosuke Iimura
  • Yoshihiro Katayama
  • Shinya Kajita
Original Article


Laccases play important roles in the development of fruiting bodies and in lignin degradation by basidiomycetes. In this study, we present novel phenotypes of transgenic tobacco plants with a chimeric gene for fungal laccase under the control of the cauliflower mosaic virus 35S promoter. At the flowering stage, the transgenic plants that produced recombinant laccase had brownish anthers instead of the greenish anthers of wild-type plants. The brownish anthers exhibited male sterility with a nondehiscent phenotype at varying frequencies. The frequency of nondehiscence depended on the temperature at which plants were cultivated and it was higher at 24°C than at 29°C. The cell wall structures of transgenic anther tissues were almost the same as in the wild type, but the stomium was severely deformed, and abnormal components were apparent in cells of the endothecium and epidermis. Furthermore, the pattern of deposition of flavonoids in the transgenic anther epidermis differed from the wild-type pattern. The expression of laccase also induced other phenotypic changes in the flowers of transgenic plants, namely, increased petal number, fused and petaloid stamens, and doubling of floral organs. These results indicate that the ectopic expression of laccase influences various aspects of flower development.

Key words

Laccase Nondehiscent anther Petaloid stamen Trametes versicolor Transgenic tobacco 


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  1. 1.
    Baldrian P (2006) Fungal laccases — occurrence and properties. FEMS Microbiol Rev 30:215–242CrossRefPubMedGoogle Scholar
  2. 2.
    Riva S (2006) Laccase: blue enzymes for green chemistry. Trend Biotech 24:219–226CrossRefGoogle Scholar
  3. 3.
    Leatham GF, Stahmann MA (1981) Studies on the laccase of Lentinus edodes: specificity, localization and association with the development of fruiting bodies. J Gen Microbiol 125:147–157Google Scholar
  4. 4.
    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 129:145–155CrossRefPubMedGoogle Scholar
  5. 5.
    Hoopes JT, Dean JFD (2004) Ferroxidase activity in a laccase-like multicopper oxidase from Liriodendron tulipifera. Plant Physiol Biochem 42:27–33CrossRefPubMedGoogle Scholar
  6. 6.
    Brown DM, Zeef LAH, Ellis J, Goodacre R, Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell 17:2281–2295CrossRefPubMedGoogle Scholar
  7. 7.
    Galuszka P, Frébortová J, Luhová L, Bilyeu KD, English JT, Frébort I (2005) Tissue localization of cytokinin dehydrogenase in maze: possible involvement of quinone species generated from plant phenolics by other enzymatic systems in the catalytic reaction. Plant Cell Physiol 46:716–728CrossRefPubMedGoogle Scholar
  8. 8.
    Pourcel L, Routaboul JM, Kerhoas L, Caboche M, Lepiniec L, Debeaujon I (2005) TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat. Plant Cell 17:2966–2980CrossRefPubMedGoogle Scholar
  9. 9.
    McCaig BC, Meagher RB, Dean JFD (2005) Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana. Planta 221:619–636CrossRefPubMedGoogle Scholar
  10. 10.
    Liang MX, Haroldsen V, Cai XN, Wu YJ (2006) Expression of a putative laccase gene, ZmLAC1, in maize primary roots under stress. Plant Cell Environ 29:746–753CrossRefPubMedGoogle Scholar
  11. 11.
    Wang GD, Li QJ, Luo B, Chen XY (2004) Ex planta phytoremediation of trichlorophenol and phenolic allelochemicals via an engineered secretory laccase. Nat Biotechnol 22:893–897CrossRefPubMedGoogle Scholar
  12. 12.
    Dean JF, LaFayette PR, Rugh C, Tristram AH, Hoopes JT, Eriksson KE, Merkle SA (1998) Laccases associated with lignifying vascular tissues. In: Lewis NG, Sarkanen S (eds) Lignin and lignan biosynthesis. ACS Symposium Series, American Chemical Society, Washington, DC, 697:96–108CrossRefGoogle Scholar
  13. 13.
    Wang J, Wang C, Zhu M, Yu Y, Zhang Y, Wei Z (2008) Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene. Plant Cell Tissue Organ Cult 93:303–310CrossRefGoogle Scholar
  14. 14.
    Liang MX, Davis E, Gardner D, Cai XN, Wu YJ (2006) Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 224:1185–1196CrossRefPubMedGoogle Scholar
  15. 15.
    Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA (2003) Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotech J 1:129–140CrossRefGoogle Scholar
  16. 16.
    de Wilde C, Uzan, E, Zhou Z, Kruus K, Andberg M, Buchert J, Record E, Asther M, Lomascolo A (2008) Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases. Transgenic Res 17:515–527CrossRefPubMedGoogle Scholar
  17. 17.
    Hirai H, Kashima Y, Hayashi K, Sugiura T, Yamagishi K, Kawagishi H, Nishida T (2008) Efficient expression of laccase gene from white-rot fungus Schizophyllum commune in a transgenic tobacco plant. FEMS Microbiol Lett 286:130–135CrossRefPubMedGoogle Scholar
  18. 18.
    Sonoki T, Kajita S, Ikeda S, Uesugi M, Tatsumi K, Katayama Y, Iimura Y (2005) Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants. Appl Microbiol Biotech 67:138–142CrossRefGoogle Scholar
  19. 19.
    Eggert C, Temp U, Eriksson KE (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62: 1151–1158PubMedGoogle Scholar
  20. 20.
    Dence CW (1992) The determination of lignin. In: Lin SY, Dence CW (eds) Methods in lignin chemistry. Springer, Berlin Heidelberg New York, pp 33–61Google Scholar
  21. 21.
    Casler MD, Hatfield RD (2006) Cell wall composition of smooth bromegrass plants selected for divergent fiber concentration. J Agric Food Chem 54:8206–8211CrossRefPubMedGoogle Scholar
  22. 22.
    Giusti MM, Wrolstad RE (2001) Characterization and measurement of anthocyanins by UV-visible spectroscopy. Curr Protocol Food Anal Chem (2001) F1.2.1–F1.2.13Google Scholar
  23. 23.
    Chaffey N (2002) Wood microscopical techniques. In Chaffey N (ed) Wood formation in trees. Taylor & Francis, London, pp 17–40CrossRefGoogle Scholar
  24. 24.
    Begum S, Nakaba S, Bayramzadeh V, Oribe Y, Kubo T, Funada R (2008) Temperature responses of cambial reactivation and xylem differentiation in hybrid poplar (Populus sieboldii × P. grandidentata) under natural conditions. Tree Physiol 28:1813–1819PubMedGoogle Scholar
  25. 25.
    Aastrup S, Outtrup H, Erdal K (1984) Localization of the proanthocyanidins in the barley grain. Carlsberg Res Commun 49:105–109CrossRefGoogle Scholar
  26. 26.
    Beals TP, Goldberg RB (1997) A novel ablation strategy blocks tobacco anther dehiscence. Plant Cell 9:1527–1545CrossRefPubMedGoogle Scholar
  27. 27.
    Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M (2005) The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17:2993–3006CrossRefPubMedGoogle Scholar
  28. 28.
    Yasuor H, Abu-Abied M, Belausov E, Madmony A, Sadot E, Riov J, Rubin B (2006) Glyphosate-induced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation. Plant Physiol 141:1306–1315CrossRefPubMedGoogle Scholar
  29. 29.
    Deluc L, Barrieu F, Marchive C, Lauvergeat V, Decendit A, Richard T, Carde JP, Mérillon JM, Hamdi S (2006) Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol 140:499–511CrossRefPubMedGoogle Scholar
  30. 30.
    Steiner-Lange S, Unte US, Eckstein L, Yang C, Wilson ZA, Schmelzer E, Dekker K, Saedler H (2003) Disruption of Arabidopsis thaliana MYB26 results in male sterility due to nondehiscent anthers. Plant J 34:519–528CrossRefPubMedGoogle Scholar
  31. 31.
    Yang CY, Xu ZY, Song J, Conner K, Barrena GV, Wilson ZA (2007) Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence. Plant Cell 19:534–548CrossRefPubMedGoogle Scholar
  32. 32.
    Hopp W, Seitz HU (1987) The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota. Planta 170:74–85CrossRefGoogle Scholar
  33. 33.
    Debeaujon I, Peeters AJM, Léon-Kloosterziel KM, Koornneef M (2001) The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell 13:853–871CrossRefPubMedGoogle Scholar
  34. 34.
    Kitamura S, Shikazono N, Tanaka A (2004) TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J 37:104–114CrossRefPubMedGoogle Scholar
  35. 35.
    Itoh N, Katsube Y, Yamamoto K, Nakajima N, Yoshida K (2007) Laccase-catalyzed conversion of green tea catechins in the presence of gallic acid to epitheaflagallin and epitheaflagallin 3-O-gallate. Tetrahedron 63:9488–9492CrossRefGoogle Scholar
  36. 36.
    Ghidouche S, Es-Safi NE, Ducrot PH (2008) Mechanistic study on the enzymatic oxidation of flavonols. Tetrahedron Lett 49: 619–623CrossRefGoogle Scholar
  37. 37.
    Lekounougou S, Mounguengui S, Dumarpy S, Rose C, Courty PE, Garbaye J, Gérardin P, Jacquot JP, Gelhaye E (2008) Initial stages of Fagus sylvatica wood colonization by the white-rot basidiomycete Trametes versicolor: enzymatic characterization. Int Biodet Biodeg 61:287–293CrossRefGoogle Scholar
  38. 38.
    Luo YH, Zuo Y, Su YQ, Ma HL (2008) Study on kinetic behaviors of the biocatalysis of laccase on oxidation of phenolic compounds — using catechol and epicatechin as model substrates. Chem Ind Forest Prod 28:13–17Google Scholar
  39. 39.
    Derksen J, van Wezel R, Knuiman B, Ylstra B, van Tunen AJ (1999) Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption. Planta 207:575–581CrossRefGoogle Scholar
  40. 40.
    Pourcel L, Routaboul JM, Cheynier V, Lepiniec L, Debeaujon I (2007) Flavonoid oxidation in plants: from biochemical properties to physiological functions. Trend Plant Sci 12:29–36CrossRefGoogle Scholar
  41. 41.
    Blee KA, Choi JW, O’Connell AP, Schuch W, Lewis NG, Bolwell GP (2003) A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification. Phytochemistry 64:163–176CrossRefPubMedGoogle Scholar
  42. 42.
    Mouradov A, Glassick T, Hamdorf B, Murphy L, Fowler B, Marla S, Teasdale RD (1998) NEEDLY, a Pinus radiata ortholog of FLORICAULA/LEAFY genes, expressed in both reproductive and vegetative meristems. Proc Natl Acad Sci USA 95:6537–6542CrossRefPubMedGoogle Scholar
  43. 43.
    Ahearn KP, Johnson HA, Weigel D, Wagner DR (2001) NFL1, a Nicotiana tabacum LEAFY-like gene, controls meristem initiation and floral structure. Plant Cell Physiol 42:1130–1139CrossRefPubMedGoogle Scholar
  44. 44.
    Shindo S, Sakakibara K, Sano R, Ueda K, Hasebe M (2001) Characterization of a FLORICAULA/LEAFY homologue of Gnetum parvifolium and its implications for the evolution of reproductive organs in seed plants. Int J Plant Sci 162:1199–1209CrossRefGoogle Scholar
  45. 45.
    Shiokawa T, Yamada Y, Futamura N, Osanai K, Murasugi D, Shinohara K, Kawai S, Morohoshi N, Katayama Y, Kajita S (2008) Isolation and functional analysis of the CjNdly gene, a homolog in Cryptomeria japonica of FLORICAULA/LEAFY genes. Tree Physiol 28:21–28PubMedGoogle Scholar
  46. 46.
    Tognolli M, Penel C, Greppin H, Simon P (2002) Analysis and expression of class III peroxidase large gene family in Arabidopsis thaliana. Gene 288:129–138CrossRefPubMedGoogle Scholar

Copyright information

© The Japan Wood Research Society 2010

Authors and Affiliations

  • Zannatul Nasrin
    • 1
  • Misato Yoshikawa
    • 1
  • Yuki Nakamura
    • 1
  • Shahanara Begum
    • 2
  • Satoshi Nakaba
    • 2
  • Mikiko Uesugi
    • 1
  • Yuriko Osakabe
    • 3
  • Tomonori Sonoki
    • 4
  • Kanna Sato
    • 1
  • Ryo Funada
    • 2
  • Yosuke Iimura
    • 5
  • Yoshihiro Katayama
    • 1
  • Shinya Kajita
    • 1
  1. 1.Graduate School of Bio-Applications and Systems EngineeringTokyo University of Agriculture and TechnologyKoganei, TokyoJapan
  2. 2.Graduate School of Agricultural ScienceTokyo University of Agriculture and TechnologyFuchuJapan
  3. 3.Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan
  4. 4.Faculty of Agriculture and Life ScienceHirosaki UniversityHirosakiJapan
  5. 5.Institute for Environmental Management TechnologyNational Institute of Advanced Industrial Science and TechnologyTsukubaJapan

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