Antibacterial, Antihemolytic, Cytotoxic, Anticancer, and Antileishmanial Effects of Ajuga bracteosa Transgenic Plants

Herbal and traditional medicines can play a pivotal role in combating cancer and neglected tropical diseases. Ajuga bracteosa, family Lamiaceae, is an important medicinal plant. The genetic transformation of A. bracteosa with rol genes of Agrobacterium rhizogenes further enhances its metabolic content. This study aimed at undertaking the molecular, phytochemical, and in vitro biological analysis of A. bracteosa extracts. We transformed the A. bracteosa plant with rol genes and raised the regenerants from the hairy roots. Transgenic integration and expression of rolB were confirmed by conventional polymerase chain reaction (PCR) and qPCR analysis. The methanol: chloroform crude extracts of wild-type plants and transgenic regenerants were screened for in vitro antibacterial, antihemolytic, cytotoxic, anticancer, and leishmanial activity. Among all plants, transgenic line 3 (ABRL3) showed the highest expression of the rolB gene. Fourier transform infra-red (FTIR) analysis confirmed the enhanced number of functional groups of active compounds in all transgenic lines. Moreover, ABRL3 exhibited the highest antibacterial activity, minimum hemolytic activity (CC50 = 7293.05 ± 7 μg/mL) and maximum antileishmanial activity (IC50 of 56.16 ± 2 μg/mL). ABRL1 demonstrated the most prominent brine shrimp cytotoxicity (LD5039.6 ± 4 μg/mL). ABRL3 was most effective against various human cancer cell lines with an IC50 of 57.1 ± 2.2 μg/mL, 46.2 ± 1.1 μg/mL, 72.4 ± 1.3 μg/mL, 73.3 ± 2.1 μg/mL, 98.7 ± 1.6 μg/mL, and 97.1 ± 2.5 μg/mL against HepG2, LM3, A549, HT29, MCF-7, and MDA-MB-231, respectively. Overall, these transgenic extracts may offer a cheaper therapeutic source than the more expensive synthetic drugs.

Polyphenol Rich Ajuga bracteosa Transgenic Regenerants Display Better Pharmacological Potential

Ajuga bracteosa Wall. ex Benth. is an endangered medicinal herb traditionally used against different ailments. The present study aimed to create new insight into the fundamental mechanisms of genetic transformation and the biological activities of this plant. We transformed the A. bracteosa plant with rol genes of Agrobacterium rhizogenes and raised the regenerants from the hairy roots. These transgenic regenerants were screened for in vitro antioxidant activities, a range of in vivo assays, elemental analysis, polyphenol content, and different phytochemicals found through HPLC. Among 18 polyphenolic standards, kaempferol was most abundant in all transgenic lines. Furthermore, transgenic line 3 (ABRL3) showed maximum phenolics and flavonoids content among all tested plant extracts. ABRL3 also demonstrated the highest total antioxidant capacity (8.16 ± 1 μg AAE/mg), total reducing power, (6.60 ± 1.17 μg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 μg/mL), hydroxyl ion scavenging (IC50 = 122.5 ± 0.90 μg/mL), and iron-chelating power (IC50 = 154.8 ± 2 μg/mL). Moreover, transformed plant extracts produced significant analgesic, anti-inflammatory, anticoagulant, and antidepressant activities in BALB/c mice models. In conclusion, transgenic regenerants of A. bracteosa pose better antioxidant and pharmacological properties under the effect of rol genes as compared to wild-type plants.

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.

Influence of some rol genes on sugar content in Nicotiana and Vaccinium

In natural conditions, insertion of Agrobacterium T-DNA into the plant genome and its subsequent transfer via sexual reproduction has been shown for several dozens of species, including species from genera Nicotiana and Vaccinium. In the framework of investigation of possible function of cT-DNA in naturally transgenic species we have shown, that increasing of expression of rolC in Nicotiana tabacum is associated with increase of amount of glucose and total sugar content. Similar trend was observed for rolB/C-like gene in Vaccinium.

The Health Promoting Bioactivities of Lactuca sativa can be Enhanced by Genetic Modulation of Plant Secondary Metabolites

Plant secondary metabolites are protective dietary constituents and rol genes evidently increase the synthesis of these versatile phytochemicals. This study subjected a globally important vegetable, lettuce (Lactuca sativa) to a combination of untargeted metabolomics (LC-QTof-MS) and in vitro bioactivity assays. Specifically, we examined the differences between untransformed cultured lettuce (UnT), lettuce transformed with either rolABC (RA) or rolC (RC) and commercially grown (COM) lettuce. Of the 5333 metabolite features aligned, deconvoluted and quantified 3637, 1792 and 3737 significantly differed in RA, RC and COM, respectively, compared with UnT. In all cases the number of downregulated metabolites exceeded the number increased. In vitro bioactivity assays showed that RA and RC (but not COM) significantly improved the ability of L. sativa to inhibit α-glucosidase, inhibit dipeptidyl peptidase-4 (DPP-4) and stimulate GLP-1 secretion. We putatively identified 76 lettuce metabolites (sesquiterpene lactones, non-phenolic and phenolic compounds) some of which were altered by several thousand percent in RA and RC. Ferulic acid levels increased 3033–9777%, aminooxononanoic acid increased 1141–1803% and 2,3,5,4′tetrahydroxystilbene-2-O-β-d-glucoside increased 40,272–48,008%. Compound activities were confirmed using commercially obtained standards. In conclusion, rol gene transformation significantly alters the metabolome of L.sativa and enhances its antidiabetic properties. There is considerable potential to exploit rol genes to modulate secondary metabolite production for the development of novel functional foods. This investigation serves as a new paradigm whereby genetic manipulation, metabolomic analysis and bioactivity techniques can be combined to enable the discovery of novel natural bioactives and determine the functional significance of plant metabolites.