Open Access
Issue
BIO Web Conf.
Volume 18, 2020
IV All-Russian Plant Protection Congress with international participation “Phytosanitary Technologies in Ensuring Independence and Competitiveness of the Agricultural Sector of Russia”
Article Number 00020
Number of page(s) 4
DOI https://doi.org/10.1051/bioconf/20201800020
Published online 06 March 2020

© The Authors, published by EDP Sciences, 2020

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 Introduction

Agrobacterium-mediated transformation is the key method of genetic engineering of plants aimed to introduce novel genes into their genomes. At the same time in natural conditions, horizontal transfer of T-DNA genes into the plant genomes has been shown for several dozens of species, including species from genera Nicotiana and Vaccinium [1-4]. Such T-DNA was termed as «cellular T-DNA» (cT-DNA). One of the most conserved genes in cT-DNA is rolC. Its expression has been shown in Nicotiana and Linaria [5-6]. It is related somehow to sugar content [7]. Gene rolC belongs to the plast genes family [8]. The plast genes are identified by their common ancestry. They are mostly described in T-DNAs. The plast genes can modify plant growth in different ways, but the molecular basis of their function remains largely unknown. Just a few agrobacterial plast genes, including rolB, rolC, orf13 and 6b, have been studied in detail. The list of plast genes was significantly expanded due to the discovery of new types of cT-DNA. Some plast genes have a significant potential for applied biology, agriculture and may be used to modify the growth of crops [8]. It is believed that the natural transformation of sweet potato with plast genes contributed to its domestication [3]. An interesting plast gene resembling rolB and rolC features was found in Vaccinium macrocarpon [4]. It is worth noting that all previously studied by us samples of V. macrocarpon contain intact gene while all samples of V. oxycoccos contain a deletion within the plast gene. Nicotiana tabacum contains intact rolC. Transgenic lines, containing an additional copy of rolC under the control of a dexamethasone-inducible promoter, were obtained based on Samsun cultivar of N. tabacum. It is possible to increase gene expression (as compared with the control) by adding the inductor to the cultural medium. Previously, the total sugar content and transport were studied in such plants [7].

In this study we compared the carbohydrates patterns in tobacco plants, differing in expression of the rolC gene and in Vaccinium plants, carrying intact plast gene and deleted one.

2 Materials and methods

Plant material was represented by 2 lines of cranberries: line GVo of Vaccinium oxycoccos L. and line GVm of Vaccinium macrocarpon Aiton; and three genotypes of tobacco: N. tabacum, cv. Samsun and two transgenic lines, derived from this cultivar and containing rolC of different origin under Pdex: A. rhizogenes rol C (Pdex-A4-rolC) and N. tabacum rol C (Pdex-trolC) correspondingly [7].

Plants of genera Vaccinium and Nicotiana were cultivated in aseptic culture on MS medium at 24h photoperiod at 22°C. Transgenic N. tabacum plants, containing rol C under dexamethasone-inducible promoter and control non-transgenic plants were cultivated for 7 days on MS media supplied with 10 μM of dexamethasone or on MS0 media [9]. Shoots were ground in liquid nitrogen, ethanol-extracted (80°C, 30 min) and analyzed by HPTLC (high-performance thin-layer chromatography). The sugar standards and extracted samples were processed on the automated HPTLC system (CAMAG, Muttenz, Switzerland). The TLC plate was developed in chloroform-acetone-methanol-acetic acid-H2O 50:20:10:10:5 v/v. The developed plate was carried out with the aniline reagent in methanol under heating. All tracks in the plate were scanned at 454 nm wavelength and individual Rf values of peaks were obtained. These data were matched with the standard. The experiment was done in three replicates. Statistical processing was performed by analysis of variance (ANOVA).

3 Results and discussion

Samples of Vaccinium and Nicotiana differed in total carbohydrate content and ratio. ANOVA have shown statistically significant differences in total carbohydrate content, glucose and sucrose composition among samples of Vaccinium (p≤0.05) (fig. 1A). Analysing N. tabacum lines we have shown statistically significant effect (p≤0.05) of both studied factors (genotype and cultural medium composition) on the sugar content (fig 1B). Difference in sugar levels of three studied tobacco genotypes without dexamethasone treatment can be partly explained by the effect of the localization of T-DNA in the genomes. In addition, dexamethasone itself increases sugar content of tobacco plants. However, the portion of the influence of dexamethasone on the level of sugars is higher in transgenic lines. For the Pdex-trolC line, Fischer intra-class correlation coefficient is 74%, for the Pdex-A4rolC line it is 49%, and in non-transformed plants it is only 17%. The carbohydrates patterns in tobacco plants, differing in expression of the rolC gene and in Vaccinium plants, carrying intact plast gene and deleted one, have shown some common features. Firstly, the sugar levels in the induced transgenic N. tabacum (Pdex-trolC and Pdex-A4rolC) plants were higher than in N. tabacum cv. Samsun and transgenic plants without induction of rolC expression (p≤0.05). In V. macrocarpon total sugar content is higher, than in V. oxycoccos. Secondly, extracts from induced transgenic plant and extracts from V. macrocarpon show an increased level of glucose comparing to control plants. Earlier in the literature there was an idea, that the products of the rolC and rolB genes are beta-glucosidases [11-12]. However, later it was criticized [8]. Our results can be explained by an increase of glucosidase activity in case of the expression of rolC and rolB/C- like gene. However, we cannot say whether this is a direct effect of studied genes, or an indirect one.

This study was supported by a grant to TM from the RFBR 18-016-00118. Transgenic seeds [7] were kindly represented by Prof. L. Otten (IBMP, France).

thumbnail Fig. 1.

Sugar content of Vaccinium (A) and Nicotiana (B) plants. Significant difference (p≤0.05) for total sugar content is shown by *, for glucose and sucrose - ⚫

References

All Figures

thumbnail Fig. 1.

Sugar content of Vaccinium (A) and Nicotiana (B) plants. Significant difference (p≤0.05) for total sugar content is shown by *, for glucose and sucrose - ⚫

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