Transformed Tissue of Dionaea Muscipula J. Ellis As a Source of Biologically Active Phenolic Compounds with Bactericidal Properties
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Applied Microbiology and Biotechnology (2021) 105:1215–1226 https://doi.org/10.1007/s00253-021-11101-8 APPLIED MICROBIAL AND CELL PHYSIOLOGY Transformed tissue of Dionaea muscipula J. Ellis as a source of biologically active phenolic compounds with bactericidal properties Wojciech Makowski1 & Aleksandra Królicka2 & Anna Nowicka3,4 & Jana Zwyrtková3 & Barbara Tokarz1 & Ales Pecinka3 & Rafał Banasiuk5 & Krzysztof Michał Tokarz1 Received: 14 August 2020 /Revised: 2 December 2020 /Accepted: 5 January 2021 / Published online: 15 January 2021 # The Author(s) 2021 Abstract The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4- naphtoquinone derivative, plumbagin (up to 106.63 mg × g−1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis,andEscherichia coli andupto7%againstPseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. Key points • Rhizobium-mediated transformation created Dionaea muscipula teratomas. • Transformed plants had highly increased synthesis of phenolic compounds. • The MBC value was connected with plumbagin and phenolic acid concentrations. Keywords Phenolic acids . Plumbagin . Rhizobium rhizogenes . Teratomas . Venus flytrap * Wojciech Makowski 2 Intercollegiate Faculty of Biotechnology UG and MUG, Laboratory [email protected] of Biologically Active Compounds, University of Gdansk, Gdansk, Poland * Aleksandra Królicka [email protected] 3 Institute of Experimental Botany, Czech Acad Sci, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic 1 Department of Botany, Physiology and Plant Protection, Faculty of 4 Biotechnology and Horticulture, University of Agriculture in The Franciszek Górski Institute of Plant Physiology, The Polish Krakow, Krakow, Poland Academy of Sciences, Krakow, Poland 5 Institute of Biotechnology and Molecular Medicine, Gdansk, Poland 1216 Appl Microbiol Biotechnol (2021) 105:1215–1226 Introduction commonly used sources of plumbagin, like Plumbago plants (Makowski et al. 2020). Rhizobium rhizogenes (former: Agrobacterium rhizogenes)is Since plants from the family Droseraceae are endangered bacteria from the family Rhizobiaceae and is a “natural genet- species and exploitation of natural habitats is impossible, ic engineer” because of the ability to transfer T-DNA (transfer in vitro propagation protocols were established (Banasiuk DNA) and incorporate bacteria-derived genes into the plant et al. 2012). Therefore, the implementation of biotechnologi- genome (Georgiev et al. 2007). The natural features of this cal tools for the enhancement of secondary metabolite produc- Gram-negative soil bacterium are used in the plant biotech- tion, like elicitation (Putalun et al. 2010; Boonsnongcheepa nology for obtaining transformed organisms with new unique et al. 2019) or genetic transformation became possible properties. During T-DNA transfer to plant cells, (Królicka et al. 2010). Nevertheless, increasing valuable phy- R. rhizogenes pass on the set of rol genes occurring in Ri tochemicals with elicitation and obtaining transformed plants plasmids (root-inducing plasmids). These genes encode spe- is a difficult task because of carnivorous plant biology cific proteins responsible for control over auxin and cytokinin (Blehova et al. 2015;Makowskietal.2019). synthesis in plant cells (Guillon et al. 2006). Usually, the The main goals of the presented study were (i) transforma- consequence of R. rhizogenes infection is the change in plant tion of the medicinal plant D. muscipula using wild strains of hormone balance and rise of the hairy root phenotype. R. rhizogenes bacteria, (ii) with simultaneously increased syn- However, depending on the plant genotype, transformed thesis of phenolic compounds, in particular plumbagin, and shoots (teratomas) may occur instead of hairy roots (iii) evaluation of bactericidal properties of extracts derived (Królicka et al. 2010). Despite the infection mechanism of from transformed plants against clinical strains of pathogenic plants by R. rhizogenes being known for a few decades, the bacteria. We hypothesized that inoculation of the Venus physiological consequences of such events related to plant flytrap with Rhizobium bacteria would incorporate rol genes species need to be studied. into plant DNA. The purpose was to establish transformed Because of the increasing demand for plant-derived phyto- clones characterized by fast growth and high productivity of chemicals and the consumption of herbal medicines (Canter valuable secondary metabolites with strong biological et al. 2005), genetic transformation found application in the activity. field of medicinal plants (Niazian 2019). Hairy roots or tera- tomas of medicinal plants characterized by a fast growth rate and biochemical stability are promising sources of plant sec- Materials and methods ondary metabolites in large-scale propagation (Georgiev et al. 2007). Moreover, they can serve as a scientific model for Plant materials and bacterial strains used for studies on secondary metabolism engineering and the over- transformation production of phytochemicals of interests in plant tissue cul- ture (Tusevski et al. 2017). To the best of our knowledge, D. muscipula plants were propagated in in vitro conditions, many Rhizobium-mediated transformations were successfully according to Makowski et al. (2019). Whole plant tissue cul- established in medicinal plants (Królicka et al. 2001; tures were cultivated on ½ MS medium (Murashige and Gangopadhyay et al. 2010; Libik-Konieczny et al. 2020), Skoog 1962) with no growth regulators, 3% sucrose, and while transformation protocol for carnivorous plant Dionaea pH 5.5 (adjusted before autoclaving), solidified with 0.75% muscipula J. Ellis (Venus flytrap) is still missing. of agar. Conditions included a temperature of 23 ± 1 °C, fluo- Carnivorous plants belonging to the family Droseraceae rescence light of 80 μmol × m−2 ×s−1 photosynthetic photon have been used in natural medicine for centuries (Królicka flux density (PPFD), and a photoperiod of 16 h/8 h light/dark et al. 2010). The healing properties of these plants arise from cycle. Plants were subcultured in 30-day intervals. the ability to produce large amounts of phenolic compounds Wild R. rhizogenes strains, including LBA 9402 (NCPPB with strong biological activity (Gaascht et al. 2013). Extracts 1855), ATCC 15834, and A4 (ATCC 31798) were obtained from carnivorous plant tissues were proven to have antibacterial from the Laboratory of Biologically Active Compounds, (Krolicka et al. 2008;Makowskietal.2020), antioxidative University of Gdansk, Poland. Bacteria were grown on yeast (Królicka et al. 2009;Makowskietal.2020), antifungal extract beef (YEB) agar medium with 200 μMof (Padhye et al. 2010), and anticancer properties (Kawiak et al. acetosyringone (Sigma) at 26 °C in the dark. For plant trans- 2019). Moreover, previous phytochemical studies showed that formation, 48-h bacterial cultures were used. the species most abundant in phenolic derivatives in the family Droseraceae is D. muscipula (Gaascht et al. 2013). The major Transformation of D. muscipula plants 1,4-naphtoquinone derivative in the biochemical composition of the Venus flytrap is plumbagin. Furthermore, concentrations Young (4-week-old) Venus flytrap rhizomes (150 pieces) of this metabolite in D. muscipula tissue are higher than in other were inoculated for each R. rhizogenes bacteria strain. Appl Microbiol Biotechnol (2021) 105:1215–1226 1217 Inoculation was performed with preparation needle, according labeled using PCR with biotin-dUTP. Plant genomic DNA to Królicka et al. (2010). After inoculation, rhizomes were was isolated as described in the previous section. Two micro- subcultured to ½ MS medium supplemented with 3% sucrose grams DNA was digested overnight at 37 °C with 1 unit of and 0.8% of agar with pH 5.5 and grown for 3 days in the BamHI enzyme (Thermo Fisher Scientific, Waltham, MA, dark. Next, co-cultures were transferred to ½ MS medium USA). Subsequently, the samples were electrophoretically supplemented with antibiotics, claforan and carbenicillin separated overnight on 1.2% 2-amino-2-(hydroxymethyl)- (500 mg × L−1 each), to eliminate R. rhizogenes. After 4 weeks 1,3-propanediol (TRIS)–borate–EDTA agarose gels, of cultivation in the dark,