Induced expression of rolC for study of its effect on the expression of genes associated with nicotine synthesis in tobacco

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Abstract


Background. Agrobacterium rhizogenes rol genes cause not only hairy root syndrome in plants, but also affect their secondary metabolism. There are cases of increasing of nicotine content in transgenic tobacco roots expressing rolC alone or in combination with other rol genes. In this work, we evaluated the change in the expression of nicotine synthesis genes and their regulators in response to the induction of expression of rolC.

Materials and methods. Plant material was represented by three Nicotiana tabacum genotypes: cv. Samsun and two transgenic lines, derived from this cultivar and containing rolC under dexamethasone inducible promoter: A. rhizogenes rolC (Pdex-A4rolC) and N. tabacum rolC (Pdex-trolC) correspondingly. Fluidigm Biomark RT-PCR was used for evaluation of expression of QPT1, QPT2, A622, ODC, ADC, PMT1, PMT2, PMT3, PMT4, MPO1, MPO2, BBL, MATE1, MATE2, ARF6, ERF168, ERF189, A4rolC, NtrolC, and reference gene gapdh. HPLC-MS / MS analysis was used to determine content of nicotine and its derivatives in plant tissues.

Results. Expression of PMT genes for the synthesis of the pyrrolidine ring, as well as the genes, controlling enzyme for final stages of nicotine synthesis, was higher in transgenic lines without induction of rolC expression. Regulatory genes were activated by dexamethasone in both transgenic and control lines, indicating the inapplicability of rolC dexamethasone induction for their study. The level of expression of PMT and MPO genes increased over time in transgenic dexamethasone-induced lines. Nicotine content decreased in transgenic dexamethasone-induced plants.

Conclusions. The rolC gene does not play a primary role in the regulation of nicotine synthesis genes. The mechanism of regulation of different nicotine biosynthesis genes and TFs varies.


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About the authors

Gita Amini

University of Tabriz

Email: gita.amini85@gmail.com

Iran, Islamic Republic of, Tabriz

PhD, Faculty of Natural Science, Department of Plant sciences

Sofia V. Sokornova

All-Russian Institute of Plant Protection

Email: svsokornova@vizr.spb.ru
ORCID iD: 0000-0001-6718-4818
SPIN-code: 3223-0513
Scopus Author ID: 57204448871

Russian Federation, Pushkin, St. Petersburg

PhD, leading researcher, Laboratory of Phytotoxicology and Biotechnology

Hanieh Mohajjel-Shoja

University of Tabriz

Email: mohajelh@yahoo.com

Iran, Islamic Republic of, Tabriz

Doctor of Plant Biology, Assistant Professor

Andrey N. Stavrianidi

Lomonosov Moscow State University

Email: stavrianidi.andrey@gmail.com
SPIN-code: 3214-0907

Russian Federation, Moscow

PhD, Assoc. Professor, Department of Analytical Chemistry

Igor A. Rodin

Lomonosov Moscow State University

Email: igorrodin@yandex.ru
SPIN-code: 2434-5903

Russian Federation, Moscow

Doctor of Chem. Sciences, Leading Researcher, Department of analytical chemistry

Tatiana V. Matveeva

Saint Petersburg State University

Author for correspondence.
Email: radishlet@gmail.com
ORCID iD: 0000-0001-8569-6665

Russian Federation, Saint Petersburg

Doctor of Biol. Sciences, Professor, Department of Genetics and Biotechnology

References

  1. White FF, Garfinkel DJ, Huffman GA, et al. Sequence homologous to Agrobacterium rhizogenes T-DNA in the genomes of uninfected plants. Nature. 1983;301(5898):348-350. https://doi.org/10.1038/301348a0.
  2. Chen K, de Borne DF, Szegedi E, Otten L. Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana. Plant J. 2014;80(4):669-682. https://doi.org/10.1111/tpj.12661.
  3. Chen K, Otten L. Natural Agrobacterium transformants: recent results and some theoretical considerations. Front Plant Sci. 2017;8: e1600. https://doi.org/10.3389/fpls.2017.01600.
  4. Matveeva TV. Agrobacterium-mediated transformation in the evolution of plants. Curr Top Microbiol Immunol. 2018;418:421-441. https://doi.org/10.1007/82_2018_80.
  5. Flores H, Pickard J, Hoy M. Production of polyacetylenes and thiophenes in heterotrophic and photosynthetic root cultures of Asteraceae. In: Lam J, Breheler H, Arnason T, Hansen L, eds. Chemistry and biology of naturally occurring acetylenes and related compounds (NOARC) bioactive molecules. Vol. 7. Elsevier, Amsterdam; 1988. Р. 233-254.
  6. Hamill JD, Parr AJ, Rhodes MJ, et al. New routes to plant secondary products. Nat Biotechnol. 1987;5(8):800-804. https://doi.org/10.1038/nbt0887-800.
  7. Rhodes MJ, Robins RJ, Hamill JD, et al. Secondary product formation using Agrobacterium rhizogenes-transformed hairy root cultures. IAPTC Newsletter. 1987;53:2-15.
  8. Matveeva TV, Sokornova SV, Lutova LA. Influence of Agrobacterium oncogenes on secondary metabolism of plants. Phytochem Rev. 2015;14(3):541-556. https://doi.org/10.1007/s11101-015-9409-1.
  9. Saitoh F, Kawasima N. The alkaloid contents of sixty Nicotiana species. Phytochemistry. 1985;24:477. https://doi.org/10.1016/S0031-9422(00)80751-7.
  10. Palazon J, Cusido RM, Roig C, et al. Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants. Plant Cell Rep. 1998;17(5):384-390. https://doi.org/10.1007/s002990050411.
  11. Zenkner FF, Margis-Pinheiro M, Cagliari A. Nicotine biosynthesis in Nicotiana: a metabolic overview. Tobacco Science. 2019;56(1):1-9. https://doi.org/10.3381/18-063.
  12. Aoyama T, Chua NH. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 1997;11(3):605-612. https://doi.org/10.1046/j.1365-313X.1997.11030605.x.
  13. Mohajjel-Shoja H, Clément B, Perot J, et al. Biological activity of the Agrobacterium rhizogenes-derived trolC gene of Nicotiana tabacum and its functional relation to other plast genes. Mol Plant Microbe Interact. 2011;24(1):44-53. https://doi.org/10.1094/MPMI-06-10-0139.
  14. Dewey R, Xie J. Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum. Phytochemistry. 2013;94:10-27. https://doi.org/10.1016/j.phytochem.2013.06.002.
  15. Shoji T, Kajikawa M, Hashimoto T. Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell. 2010;22(10): 3390-3409. https://doi.org/10.1105/tpc.110. 078543.
  16. Qin Y, Bai S, Li W, et al. Transcriptome analysis reveals key genes involved in the regulation of nicotine biosynthesis at early time points after topping in tobacco (Nicotiana tabacum L.). BMC Plant Biol. 2020;20(1):30. https://doi.org/10.1186/s12870-020-2241-9.
  17. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 1962;15(3): 473-497. https://doi.org/10.1111/j.1399-3054.1962. tb08052.x.
  18. Matveeva T, Amini G. Fluidigm BioMark diagnostic panel for analysis of the expression of Nicotiana tabacum genes, associated with alkaloid synthesis. Hairy root cultures (HRCs) based applications. Methods and Protocols. Srivastava V, Mehrotra S, Mishra S, eds. Springer; 2020. Р. 229-237.
  19. Ruprecht C, Tohge T, Fernie A, et al. Transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry. J Vis Exp. 2014;(87):51393. https://doi.org/10.3791/51393.
  20. Smyth TJ, Ramachandran VN, McGuigan A, et al. Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/electrospray ionisation mass spectrometry. Rapid Commun Mass Spectrom. 2007;21(4):557-566. https://doi.org/10.1002/rcm.2871.
  21. Analysis of variance (ANOVA). Available from: http://statsoft.ru/products/STATISTICA_Base/analysis-of-variance.php/. Active on: 01.10.2020.
  22. Li C, Teng W, Shi Q, Zhang F. Multiple signals regulate nicotine synthesis in tobacco plant. Plant Signal Behav. 2007;2(4):280-281. https://doi.org/10.4161/psb.2.4.4008.
  23. Moore I, Samalova M, Kurup S. Transactivated and chemically inducible gene expression in plants. The Plant J. 2006;45(4):651-683. https://doi.org/10.1111/j.1365-313x.2006.02660.x.
  24. Meyer AD, Ichikawa T, Meins FJ. Horizontal gene transfer: regulated expression of a tobacco homologue of the Agrobacterium rhizogenes rolC gene. Mol Gen Genet. 1995;249(3):265-273. https://doi.org/10.1007/BF00290526.
  25. Matveeva T, Berezina E, Isaeva I, et al. Influence of some rol genes on sugar content in Nicotiana and Vaccinium. BIO Web of Conferences. 2020;18:00020. https://doi.org/10.1051/bioconf/20201800020.
  26. Hidalgo Martinez D, Payyavula RS, Kudithipudi C, et al. Genetic attenuation of alkaloids and nicotine content in tobacco (Nicotiana tabacum). Planta. 2020;251(4):92. https://doi.org/10.1007/s00425-020-03387-1.

Supplementary files

Supplementary Files Action
1.
Fig. 1. Nicotine biosynthesis: aspartate oxidase (AO), quinolinatesynthase (QS), quinolinatephosphoribosyl transferase (QPT), ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT), N-methylputrescine oxidase (MPO), PIP-family oxidoreductase (A622), berberine bridge enzyme-like (BBL)

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2.
Fig. 2. Relative gene expression level of A622, PMT1, PMT3 in roots of uninduced tobacco lines

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3.
Fig. 3. Dynamics of expression of the A4rolC and NtrolC genes. The graph shows the expression levels of the studied genes, relative to the reference gene gapdh

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4.
Fig. 4. Dynamics of expression of genes of transcription factors ARF6, ERF168 and genes ADS and ODS, encoding enzymes of the initial stages of the synthesis of the pyrrolidine ring. The expression levels of genes of interest of the Samsun variety at the start of the experiment was taken as 1

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5.
Fig. 5. Dynamics of expression of genes of pyrrolidine ring synthesis enzymes. The expression level of genes or interest of the Samsun variety at the start of the experiment was taken as 1

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6.
Fig. 6. The content of alkaloids in leaf extracts of tobacco lines after dexamethasone treatment (MS-dex – plants, cultivated on MS media with dexamethasone; MSO – control plants cultivated on MS media without dexamethasone)

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Copyright (c) 2021 Amini G., Sokornova S., Mohajjel-shoja H., Stavrianidi A.N., Rodin I.A., Matveeva T.V.

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