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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
  • 17 Downloads

Abstract

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.

Keywords

Lignin Antisense CCoAOMT Birch Tobacco Transgene 

Abbreviations

CCoAOMT

Caffeoyl-CoA O-methyltransferase

MS

Murashige and Skoog medium

RT-qPCR

Real-time quantitative polymerase chain reaction

cDNA

Complementary deoxyribonucleic acid

RACE

Rapid amplification of cDNA ends

WT

Wild-type

GUS

Beta-glucuronidase

Notes

Acknowledgements

This work was supported by a grant from the Fundamental Research Funds for the Central Universities (No. 2572015EA05). We thank LetPub (www.letpub.com) 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)

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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

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