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

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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


Iran, Islamic Republic of, Tabriz

PhD, Faculty of Natural Science, Department of Plant sciences

Sofia V. Sokornova

All-Russian Institute of Plant Protection

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


Iran, Islamic Republic of, Tabriz

Doctor of Plant Biology, Assistant Professor

Andrey N. Stavrianidi

Lomonosov Moscow State University

SPIN-code: 3214-0907

Russian Federation, Moscow

PhD, Assoc. Professor, Department of Analytical Chemistry

Igor A. Rodin

Lomonosov Moscow State University

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.
ORCID iD: 0000-0001-8569-6665

Russian Federation, Saint Petersburg

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


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Supplementary files

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

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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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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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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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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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