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

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.

Homologs of the rolC gene of naturally transgenic toadflaxes Linaria vulgaris and Linaria creticola are expressed in vitro

Agrobacterium mediated transformation is the most common way for obtaining transgenic plants in laboratory conditions. At the same time, there are species inside the genera Nicotiana, Linaria and Ipomoea that contain homologs of agrobacterial T-DNA genes as a result of genetic transformation of their ancestral forms in natural conditions. Such plants are called naturally transgenic plants, and T-DNA in their genomes is called cellular (cT-DNA). It is proposed that in the evolution of these genera, the introduced sequences played an important role. This idea is confirmed by the data on the expression of some T-DNA genes in Nicotiana and Ipomoea. Until the last moment, the expression of cT-DNA genes in Linaria has not been documented. However, the analysis of the nucleotide sequence indicates the functionality of rolC gene in L. vulgaris Mill., L. acutiloba Fisch. ex Rchb., L. genistifolia (L.) Mill. In this research work, we have sequenced the rolC homolog in one more toadflax species (Linaria creticola Kuprian). The in silico analysis of this gene has shown that it can encode a full-length peptide. Using the real time RT-PCR method, we have demonstrated that the rolC homolog is expressed in vitro in shoots, roots and calli of L. vulgaris Mill., as well as in shoots of L. creticola Kuprian. The results obtained are an important argument in favor of the fact that cT-DNA is functional and that its fixation in genomes played a certain role in the evolutionary process. However, the level of expression of the gene studied is quite low. A similar trend was observed in other naturally transgenic species. This can explain the absence of explicit morphological differences of species containing cT-DNA from their non-transgenic relatives.

Study creating and cloning hairy root lines in Talinum paniculatum plants

Talinum paniculatum Gaertn. contains flavonoid and saponins that have strong antioxidant properties, which are used to treat a number of conditions such as inflammation, allergies, stomach ulcers, ... However, the amount of flavonoid synthesized naturally in Talinum paniculatum plants is very low (about 0.897 mg /g fresh leaves). Therefore a method has been proposed for enhancing flavonoid content in Talinum paniculatum plants by applying tissue culture technique to produce the hairy roots to enhance biomass. This study present the results of optimization of the gene transfer process to create the hairy root lines through Agrobacterium rhizogenes (A. rhizogenes) in Talinum paniculatum plants. Of the three types of materials that infect by A. rhizogenes (cotyledon, stem, leaf tissue), leaf tissue is a suitable material for transforming and inducing hairy roots. Density of bacteria corresponding to OD600 value=0.6; concentration AS 100 μmol/l; Infection time of 10 minutes; 2 days of co-culture; cefotaxime concentrations of 500 mg/l are suitable conditions for inducing hairy roots from leaf tissue. In state of the liquid MS medium without growth regulator, shaking culture conditions are suitable for hairy roots growth. The test result of presence of rolC gene by PCR and the absence of the virD2 gene asserted that 5 lines of hairy roots were produced from Talinum paniculatum plants.

Functional analysis of cT-DNAs in naturally transformed plants, recent findings and general considerations

Several cases have been reported of naturally transformed plant species. These plants contain cellular T-DNAs (cT-DNAs) derived from ancient infections by Agrobacterium. We have determined the structure of 4 different cT-DNAs in N. tomentosiformis, the paternal ancestor of N. tabacum, and found several intact open reading frames. Among these, TB-mas2’ and TA-rolC were tested for activity. TB-mas2’ encodes desoxyfructosylglutamine (DFG) synthesis. Some N. tabacum cultivars show very high TB-mas2’ expression and produce DFG in their roots. The TA-rolC gene is biologically active and when expressed under strong constitutive promoter control, generates growth changes in N. tabacum. Based on these first data on the structure and function of cT-DNAs I present a theoretical model on the origin and evolution of naturally transformed plants, which may serve as a basis for further research in this field.

Effect of the Number of rol Genes Integrations on Phenotypic Variation in Hairy Root-Derived Hypericum perforatum L. Plants

The extent of phenotypic variation of St. John’s wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of TL-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes