Ectopic expression of an antisense BpCCoAOMT gene from Betula platyphylla Suk. affects growth and development of tobacco due to lignin content reduction

  • Yong Zhang
  • Xiaoqing Hu
  • Yaoqiang Zheng
  • Xuemei LiuEmail author
Original Article


Lignin is an aromatic polymer and macromolecular organic material and is one of the major components of plant cellular walls. It provides mechanical support for plants and protection against pathogen invasion. Caffeoyl-CoA O-methyltransferase (CCoAOMT) is recognized to be involved in phenylpropanoid metabolism and lignin synthesis, and plays an important role in precursor synthesis of G-lignin units. In this study, a gene encoding CCoAOMT (Genbank ID: AY860952) was isolated from birch (Betula platyphylla Suk.). The predicted BpCCoAOMT polypeptide had high affinity with CCoAOMT from other species. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that BpCCoAOMT is most highly expressed in young stems. To study its function in vivo, antisense BpCCoAOMT complementary deoxyribonucleic acid (cDNA) was transformed into tobacco (SR-1) by the Agrobacterium tumefaciens-mediated method. The expression of NtCCoOAMT in antisense BpCCoAOMT transgenic tobacco was down-regulated and total lignin content of transgenic plants decreased by 39% compared to control plants. Maüle reagent was used to distinguish lilac lignin and guaiac wood lignin in situ, which enables S-lignin to exhibit a specific red response. Results revealed that all transgenic tobacco plants were dark brown, while controls were dark red. This indicated that S-lignin content in the xylem was reduced. Compared to wild-type (WT) plants, transgenic tobacco plants had delayed flowering and some had slender stems, curling, and easy lodging. This indicates that the decrease in lignin content interferes with the normal growth of plants.


Lignin Antisense CCoAOMT Birch Tobacco Transgene 



Caffeoyl-CoA O-methyltransferase


Murashige and Skoog medium


Real-time quantitative polymerase chain reaction


Complementary deoxyribonucleic acid


Rapid amplification of cDNA ends







This work was supported by a grant from the Fundamental Research Funds for the Central Universities (No. 2572015EA05). We thank LetPub ( for its linguistic assistance during the preparation of this manuscript.

Author contributions

YZ and XL designed the study. YZ and Yaoqiang Zheng performed the experiments. YZ, XH, and Yaoqiang Zheng conducted the data analyses. XL provided guidance for the study. YZ and XH wrote and revised the manuscript. All authors approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interest.

Supplementary material

13562_2019_533_MOESM1_ESM.tif (1.5 mb)
Figure S1. Transforming tobacco by antisense BpCCoAOMT. (a) Expression vector map of pBI121-antiBpCCoAOMT. BpCCoAOMT is antisense CCoAOMT. NOS is the nopaline synthase terminator. XbaI and BamHI are restriction enzymes. Arrows indicate approximate tangent positions. Diagram is not drawn to scale. (b) The callus began to form 12 days after transformation. (c) Kanamycin resistant clusters formed 20 days after transformation. (d) Thirty days after transformation of kanamycin resistant clusters. (e) Rooting status of transgenic seedlings in MS medium supplemented with kanamycin. (TIFF 1516 kb)
13562_2019_533_MOESM2_ESM.tif (1.8 mb)
Figure S2. Transgenic tobacco stained with Wiesner reagent. (a and c) Transgenic and (a and c) wild-type (b and d) tobacco were used for histochemical staining. Although the strain with the greatest reduction in lignin was selected, the staining effect remained insignificant compared to wild-type tobacco. (TIFF 1840 kb)
13562_2019_533_MOESM3_ESM.tif (55 kb)
Figure S3. Northern blot analysis of B. platyphylla CCoAOMT. Lanes 1-5 represent different sampling periods: 1) July 4th; 2) July 22nd; 3) August 4th; 4) August 18th; 5) September 9th. (TIFF 55 kb)


  1. Chapple CC, Vogt T, Ellis BE, Somerville CR (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell 4:1413–1424PubMedPubMedCentralGoogle Scholar
  2. Chen C, Meyermans H, Burggraeve B, De Rycke RM, Inoue K, De Vleesschauwer V, Steenackers M, Van Montagu MC, Engler GJ, Boerjan WA (2000) Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar. Plant Physiol 123:853–867CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen F, Srinivasa RM, Temple S, Jackson L, Shadle G, Dixon RA (2006) Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.). Plant J 48:113–124CrossRefPubMedGoogle Scholar
  4. Do CT, Pollet B, Thevenin J, Sibout R, Denoue D, Barriere Y, Lapierre C, Jouanin L (2007) Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 226:1117–1129CrossRefPubMedGoogle Scholar
  5. Guo D, 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 13:73–88CrossRefPubMedPubMedCentralGoogle Scholar
  6. Horsch R, Fry JHN, Eichholtz D, Rogers SA, Fraley R (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  7. Inoue K, Sewalt VJ, Murray GB, Ni W, Sturzer C, Dixon RA (1998) Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification. Plant Physiol 117:761–770CrossRefPubMedPubMedCentralGoogle Scholar
  8. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5:387–405CrossRefGoogle Scholar
  9. Joshi CP, Chiang VL (1998) Conserved sequence motifs in plant S-adenosyl-l-methionine-dependent methyltransferases. Plant Mol Biol 37:663–674CrossRefPubMedGoogle Scholar
  10. 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 119:153–164CrossRefPubMedPubMedCentralGoogle Scholar
  11. Li L, Osakabe Y, Joshi CP, Chiang VL (1999) Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine. Plant Mol Biol 40:555–565CrossRefPubMedGoogle Scholar
  12. Liu X, Wang Q, Chen P, Song F, Guan M, Jin L, Wang Y, Yang C (2012) Four novel cellulose synthase (CESA) genes from Birch (Betula platyphylla Suk.) involved in primary and secondary cell wall biosynthesis. Int J Mol Sci 13:12195–12212CrossRefPubMedPubMedCentralGoogle Scholar
  13. Liu X, Luo Y, Wu H, Xi W, Yu J, Zhang Q, Zhou Z (2016) Systematic analysis of O-methyltransferase gene family and identification of potential members involved in the formation of O-methylated flavonoids in Citrus. Gene 575:458–472CrossRefPubMedGoogle Scholar
  14. Ludwig CH, Sarkanen KV (1971) Lignins: occurrence, formation, structure and reactions. Wiley, New YorkGoogle Scholar
  15. Marita JM, Ralph J, Hatfield RD, Guo D, Chen F, Dixon RA (2003) Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. Phytochemistry 62:53–65CrossRefPubMedGoogle Scholar
  16. Martz F, Maury S, Pincon G, Legrand M (1998) cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme. Plant Mol Biol 36:427–437CrossRefPubMedGoogle Scholar
  17. Maury S, Geoffroy P, Legrand M (1999) Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns. Plant Physiol 121:215–224CrossRefPubMedPubMedCentralGoogle Scholar
  18. Meng H, Campbell WH (1998) Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development. Plant Mol Biol 38:513–520CrossRefPubMedGoogle Scholar
  19. Meyermans H, Morreel K, Lapierre C, Pollet B, De Bruyn A, Busson R, Herdewijn P, Devreese B, Van Beeumen J, Marita JM, Ralph J, Chen C, Burggraeve B, Van Montagu M, Messens E, Boerjan W (2000) Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J Biol Chem 275:36899–36909CrossRefGoogle Scholar
  20. Nakashima J, Chen F, Jackson L, Shadle G, Dixon RA (2008) Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types. New Phytol 179:738–750CrossRefPubMedGoogle Scholar
  21. Pang SL, Ong SS, Lee HH, Zamri Z, Kandasamy KI, Choong CY, Wickneswari R (2014) Isolation and characterization of CCoAOMT in interspecific hybrid of Acacia auriculiformis × Acacia mangium—a key gene in lignin biosynthesis. Genet Mol Res 13:7217–7238CrossRefPubMedGoogle Scholar
  22. 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 57:1167–1176CrossRefPubMedGoogle Scholar
  23. Piquemal J, Chamayou S, Nadaud I, Beckert M, Barriere Y, Mila I, Lapierre C, Rigau J, Puigdomenech P, Jauneau A, Digonnet C, Boudet AM, Goffner D, Pichon M (2002) Down-regulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol 130:1675–1685CrossRefPubMedPubMedCentralGoogle Scholar
  24. Rakoczy M, Femiak I, Alejska M, Figlerowicz M, Podkowinski J (2018) Sorghum CCoAOMT and CCoAOMT-like gene evolution, structure, expression and the role of conserved amino acids in protein activity. Mol Genet Genom 293:1077–1089CrossRefGoogle Scholar
  25. Richards E, Reichardt M, Rogers S (1997) Preparation of plant DNA using CTAB. Mol Biol 3:1210–1211Google Scholar
  26. Sambrook J, FRITSH E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Pres, New YorkGoogle Scholar
  27. Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH (2006) Regulation of Arabidopsis thaliana 4-coumarate: coenzyme-A ligase-1 expression by artificial zinc finger chimeras. Plant Biotechnol J 4:103–114CrossRefPubMedGoogle Scholar
  28. Shi R, Sun YH, Li Q, Heber S, Sederoff R, Chiang VL (2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes. Plant Cell Physiol 51:144–163CrossRefPubMedGoogle Scholar
  29. Tronchet M, Balague C, Kroj T, Jouanin L, Roby D (2010) Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Mol Plant Pathol 11:83–92CrossRefPubMedGoogle Scholar
  30. Vanholme R, Cesarino I, Rataj K, Xiao Y, Sundin L, Goeminne G, Kim H, Cross J, Morreel K, Araujo P, Welsh L, Haustraete J, McClellan C, Vanholme B, Ralph J, Simpson GG, Halpin C, Boerjan W (2013) Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341:1103–1106CrossRefPubMedGoogle Scholar
  31. Wagner A, Tobimatsu Y, Phillips L, Flint H, Torr K, Donaldson L, Pears L, Ralph J (2011) CCoAOMT suppression modifies lignin composition in Pinus radiata. Plant J 67:119–129CrossRefPubMedGoogle Scholar
  32. Walker AM, Sattler SA, Regner M, Jones JP, Ralph J, Vermerris W, Sattler SE, Kang C (2016) The structure and catalytic mechanism of sorghum bicolor Caffeoyl-CoA O-methyltransferase. Plant Physiol 172:78–92CrossRefPubMedPubMedCentralGoogle Scholar
  33. Whetten RW, MacKay JJ, Sederoff RR (1998) Recent advances in understanding lignin biosynthesis. Annu Rev Plant Physiol Plant Mol Biol 49:585–609CrossRefPubMedGoogle Scholar
  34. Whetten RW, MacKay JJ, Sederoff RR (2000) Cloning of cDNA encoding CCoAOMT from Populus tomentosa and down-regulation of lignin content in transgenic plant expressing antisense gene. Acta Botanica Sinica 49:585–609Google Scholar
  35. Widiez T, Hartman TG, Dudai N, Yan Q, Lawton M, Havkin-Frenkel D, Belanger FC (2011) Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases. Plant Mol Biol 76:475–488CrossRefPubMedGoogle Scholar
  36. Xu B, Escamilla-Trevino LL, Sathitsuksanoh N, Shen Z, Shen H, Zhang YH, Dixon RA, Zhao B (2011) Silencing of 4-coumarate: coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production. New Phytol 192:611–625CrossRefPubMedGoogle Scholar
  37. Yang XH, Li XG, Li BL, Zhang DQ (2014) Genome-wide transcriptional profiling reveals molecular signatures of secondary xylem differentiation in Populus tomentosa. Genet Mol Res 13:9489–9504CrossRefPubMedGoogle Scholar
  38. Ye ZH, Varner JE (1995) Differential expression of two O-methyltransferases in lignin biosynthesis in Zinnia elegans. Plant Physiol 108:459–467CrossRefPubMedPubMedCentralGoogle Scholar
  39. Ye ZH, Kneusel RE, Matern U, Varner JE (1994) An alternative methylation pathway in lignin biosynthesis in Zinnia. Plant Cell 6:1427–1439PubMedPubMedCentralGoogle Scholar
  40. Yokoyama R, Nishitani K (2004) Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis. Plant Cell Physiol 45:1111–1121CrossRefPubMedGoogle Scholar
  41. Zhao Q (2016) Lignification: flexibility, biosynthesis and regulation. Trends Plant Sci 21:713–721CrossRefGoogle Scholar
  42. Zhao HY, Wei JH, Song YR (2004) [Advances in research on lignin biosynthesis and its genetic engineering]. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30:361–370Google Scholar
  43. Zhong R, Morrison WI, Negrel J, Ye ZH (1998) Dual methylation pathways in lignin biosynthesis. Plant Cell 10:2033–2046CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zhong R, Morrison WR, Himmelsbach DS, Poole FN, Ye ZH (2000) Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants. Plant Physiol 124:563–578CrossRefPubMedPubMedCentralGoogle Scholar
  45. Zhou J, Lee C, Zhong R, Ye ZH (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21:248–266CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  • Yong Zhang
    • 1
  • Xiaoqing Hu
    • 1
  • Yaoqiang Zheng
    • 1
  • Xuemei Liu
    • 1
    Email author
  1. 1.School of Life ScienceNortheast Forestry UniversityHarbinPeople’s Republic of China

Personalised recommendations