Molecular Biology Reports

, Volume 40, Issue 2, pp 1979–1986 | Cite as

Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants

  • Eun Soo Seong
  • Ji Hye Yoo
  • Jae Geun Lee
  • Hee Young Kim
  • In Seong Hwang
  • Kweon Heo
  • Jae Kwang Kim
  • Jung Dae Lim
  • Erik J. Sacks
  • Chang Yeon Yu


Initially, we isolated the caffeic acid O-methyltransferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, we produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidases expression, which are related to the phenylpropanoid pathway and antioxidant activity. The MsCOMT-down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT-down-regulated transgenic plants. We also observed a morphological difference, with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway.


APX COMT Lignin Miscanthus sinensis PAL Transgenic plant 


  1. 1.
    Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen species and antioxidants: relationships in green cells. Physiol Plant 100:224–233CrossRefGoogle Scholar
  2. 2.
    Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. A Rev Plant Physiol Plant Mol Biol 50:601–639CrossRefGoogle Scholar
  3. 3.
    Besseau S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M (2007) Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19:148–162PubMedCrossRefGoogle Scholar
  4. 4.
    Bhuiyan NH, Selvaraj G, Wei Y, King J (2009) Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion. J Exp Bot 60:509–521PubMedCrossRefGoogle Scholar
  5. 5.
    Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546PubMedCrossRefGoogle Scholar
  6. 6.
    Carver TLW, Zeyen RJ, Robbins MP, Dearne GA (1992) Effects of the PAL inhibitor, AOPP, on oat, barley and wheat cell responses to appropriate and inappropriate formae specials of Erysiphe graminis DC. Physiol Mol Plant Pathol 41(1992):397–409CrossRefGoogle Scholar
  7. 7.
    Chen L, Aun CK, Dowling P, Belt J, Lehmann D, Wang ZY (2004) Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestility in tall fescue (Festuca arundinacea), L. Func Plant Biol 31:235–245CrossRefGoogle Scholar
  8. 8.
    Cochrane FC, Davin LB, Lewis NG (2004) The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms. Phytochemistry 65:1557–1564PubMedCrossRefGoogle Scholar
  9. 9.
    Ferrer J, Zubieta C, Dixon RA, Noel JP (2005) Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransferase. Plant Physiol 137:1009–1017PubMedCrossRefGoogle Scholar
  10. 10.
    Franke R, McMichael CM, Meyer K, Shirley AM, Cusumano JC, Chapple C (2000) Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J 22:223–234PubMedCrossRefGoogle Scholar
  11. 11.
    Ghimire BK, Seong ES, Kim EH, Ghimeray AK, Yu CY, Chung IM (2011) A comparative evaluation of the antioxidant activity of some medicinal plants popularly used in Nepal. J Med Plants Res 5:1884–1891Google Scholar
  12. 12.
    Gowri G, Bugos RC, Campbell WH, Maxwell CA, Dixon RA (1991) Stress responses in alfalfa (Medicago sativa L). X. Molecular cloning and expression of S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase, a key enzyme of lignin biosynthesis. Plant Physiol 97:7–14PubMedCrossRefGoogle Scholar
  13. 13.
    Guillaumie S, Goffner D, Barbier O, Martinant JP, Pichon M, Barrière Y (2008) Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants. BMC Plant Biol 8:71–87PubMedCrossRefGoogle Scholar
  14. 14.
    Guo DJ, Chen F, Inoue K, Blount JW, Dixon RA (2001) Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa. Impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell 13:73–88PubMedGoogle Scholar
  15. 15.
    Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369CrossRefGoogle Scholar
  16. 16.
    Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832PubMedCrossRefGoogle Scholar
  17. 17.
    Hoffmann L, Maury S, Pincon G, Geoffroy P, Fritig B, Legrand M (2000) Impacts of the repression of phenylpropanoid synthesis on development and disease resistance of transgenic tobacco. In: 6th International congress of plant molecular biology, Quebec, Canada, June, 2000, p 18–24Google Scholar
  18. 18.
    Hsieh LS, Hsieh YL, Yeh CS, Cheng CY, Yang CC, Lee PD (2010) Molecular characterization of a phenylalanine ammonialyase gene (BoPAL1) from Bambusa oldhamii. Mol Biol Rep 38:283–290PubMedCrossRefGoogle Scholar
  19. 19.
    Ishikawa T, Sakai K, Takeda T, Shigeoka S (1995) Cloning and expression of cDNA encoding a new type of ascorbate peroxidase from spinach. FEBS Lett 367:28–32PubMedCrossRefGoogle Scholar
  20. 20.
    Jouanin L, Goujon T, de Nadaı¨ V, Martin MT, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M, Lapierre C (2000) Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol 123:1363–1374PubMedCrossRefGoogle Scholar
  21. 21.
    Kawakami S, Matsumoto Y, Matsunaga A, Mayama S, Mizuno M (2002) Molecular cloning of ascorbate peroxidase in potato tubers and its response during storage at low temperature. Plant Sci 163:829–836CrossRefGoogle Scholar
  22. 22.
    Kim IJ, Chung WI (1998) Molecular characterization of a cytosolic ascorbate peroxidase in strawberry fruit. Plant Sci 133:69–77CrossRefGoogle Scholar
  23. 23.
    Kruger WM, Carver TLW, Zeyen RJ (2002) Effects of inhibiting phenolic biosynthesis on penetration resistance of barley isolines containing seven powdery mildew resistance genes or alleles. Physiol Mol Plant Pathol 61:41–51Google Scholar
  24. 24.
    Lewis NG, Yamamoto E (1990) Lignin: occurrence, biogenesis, and biodegradation. A Rev Plant Physiol Plant Mol Biol 41:455–496CrossRefGoogle Scholar
  25. 25.
    Li L, Popko JL, Umezawa T, Chiang VL (2000) 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Biol Chem 275:6537–6545PubMedCrossRefGoogle Scholar
  26. 26.
    Li X, Weng JK, Chapple C (2008) Improvement of biomass through lignin modification. Plant J 54:569–581PubMedCrossRefGoogle Scholar
  27. 27.
    Louie GV, Bowman ME, Tu Y, Mouradov A, Spangenberg G, Noel GP (2010) Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference. Plant Cell 22:4114–4127PubMedCrossRefGoogle Scholar
  28. 28.
    Ma QH, Xu Y (2008) Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis. Biochimie 90:515–524PubMedCrossRefGoogle Scholar
  29. 29.
    Marita JM, Ralph J, Hatfield RD, Chapple C (1999) NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc Natl Acad Sci USA 96:12328–12332PubMedCrossRefGoogle Scholar
  30. 30.
    Mehdy MC, Sharma YK, Sathasivan K, Bays NW (1996) The role of activated oxygen species in plant disease resistance. Plant Physiol 98:365–374Google Scholar
  31. 31.
    Millar DJ, Long M, Donovan G, Fraser PD, Boudet AM, Danoun S, Bramley PM, Bolwell GP (2007) Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids. Phytochemistry 68:1497–1509PubMedCrossRefGoogle Scholar
  32. 32.
    Mittler R, Zilinskas BA (1991) Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase. FEBS Lett 289:257–259PubMedCrossRefGoogle Scholar
  33. 33.
    Ni W, Sewalt VJH, Korth KL, Blount JW, Balance GM, Dixon RA (1996) Stress responses in alfalfa. XXI. Activation of caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase genes does not contribute to changes in metabolite accumulation in elicitor-treated cell-suspension cultures. Plant Physiol 112:717–726PubMedGoogle Scholar
  34. 34.
    Olsen KM, Lea US, Slimestad R, Verheul M, Lillo C (2008) Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis. J Plant Physiol 165:1491–1499PubMedCrossRefGoogle Scholar
  35. 35.
    Parvathi K, Chen F, Guo DJ, Blount JW, Dixon RA (2001) Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant J 25:193–202PubMedCrossRefGoogle Scholar
  36. 36.
    Pincon G, Maury S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M (2001) Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry 57:1167–1176PubMedCrossRefGoogle Scholar
  37. 37.
    Piquemal J, Chamayou S, Nadaud I, Beckert M, Barriere Y (2002) Down-regulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol 130:1675–1685PubMedCrossRefGoogle Scholar
  38. 38.
    Rae AL, Manners JM, Jones RJ, McIntyre CL, Lu DY (2001) Antisense suppression of the lignin biosynthetic enzyme, caffeate O-methyltransferase, improves in vitro digestibility of the tropical pasture legume, Stylosanthes humilis. Aust J Plant Physiol 28:289–297Google Scholar
  39. 39.
    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
  40. 40.
    Ralph J, Lapierre C, Lu F, Marita JM, Pilate G, Van Doorsselaere J, Boerjan W, Jouanin L (2001) NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase deficient poplars. J Agric Food Chem 49:86–91PubMedCrossRefGoogle Scholar
  41. 41.
    Romero I, Teresa Sanchez-Ballesta M, Maldonado R, Escribano MI, Merodio C (2008) Anthocyanin, antioxidant activity and stress-induced gene expression in high CO2-treated table grapes stored at low temperature. J Plant Physiol 165:522–530PubMedCrossRefGoogle Scholar
  42. 42.
    Shure M, Wessler S, Fedoroff N (1983) Molecular identification and isolation of the Waxy locus in Maize. Cell 35:225–233PubMedCrossRefGoogle Scholar
  43. 43.
    Song J, Wang Z (2008) Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Mol Biol Rep 36:939–952PubMedCrossRefGoogle Scholar
  44. 44.
    Suzuki S, Suzuki Y, Yamamoto N, Hattori T, Sakamoto M, Umezawa T (2009) High-throughput determination of thioglycolic acid lignin from rice. Plant Biotechnol 26:337–340CrossRefGoogle Scholar
  45. 45.
    Taga MS, Miller EE, Pratt DE (1984) Chia seeds as a source of natural lipids antioxidants. J Am Oil Chem Soc 61:928–993CrossRefGoogle Scholar
  46. 46.
    Tu Y, Rochfort S, Liu Z, Ran Y, Griffith M, Badenhorst P, Louie GV, Bowman ME, Smith KF, Noel JP, Mouradov A, Spangenberg G (2010) Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne) W. Plant Cell 22:3357–3373PubMedCrossRefGoogle Scholar
  47. 47.
    Vanholme R, Morreel K, Ralph J, Boerjan W (2008) Lignin engineering. Curr Opin Plant Biol 11:278–285PubMedCrossRefGoogle Scholar
  48. 48.
    Whetten R, Sederoff R (1995) Lignin biosynthesis. Plant Cell 7:1001–1013PubMedGoogle Scholar
  49. 49.
    Yi SY, Yu SH, Choi D (1999) Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection. Mol Cells 30:320–325Google Scholar
  50. 50.
    Yoshimura K, Yabuta Y, Ishikawa T, Shigeoka S (2000) Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stress. Plant Physiol 123:223–233PubMedCrossRefGoogle Scholar
  51. 51.
    Zhong RQ, Taylor JJ, Ye ZH (1997) Disruption of interfascicular fiber differentiation in an Arabidopsis mutant. Plant Cell 9:2159–2170PubMedGoogle Scholar
  52. 52.
    Zhou JM, Gold ND, Martin VJ, Wollenweber E, Ibrahim RK (2006) Sequential O-methylation of tricetin by a single gene product in wheat. Biochim Biophys Acta 1760:1115–1124PubMedCrossRefGoogle Scholar
  53. 53.
    Zubieta C, Kota P, Ferrer JL, Dixon RA, Noel JP (2002) Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase. Plant Cell 14:1265–1277PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Eun Soo Seong
    • 1
  • Ji Hye Yoo
    • 2
  • Jae Geun Lee
    • 2
  • Hee Young Kim
    • 2
  • In Seong Hwang
    • 2
  • Kweon Heo
    • 2
  • Jae Kwang Kim
    • 3
  • Jung Dae Lim
    • 4
  • Erik J. Sacks
    • 5
  • Chang Yeon Yu
    • 2
  1. 1.Bioherb Research InstituteKangwon National UniversityChuncheonSouth Korea
  2. 2.Department of Applied Plant Sciences, College of Agriculture and Life ScienceKangwon National UniversityChuncheonSouth Korea
  3. 3.National Academy of Agricultural ScienceRural Development AdministrationSuwonSouth Korea
  4. 4.Department of Herbal Medicine ResourceKangwon National UniversitySamcheokSouth Korea
  5. 5.Department of Crop Sciences 1101 1GB, MC-195 1206 W. Gregory Dr. UrbanaUniversity of IllinoisUrbanaUSA

Personalised recommendations