Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene.

BACKGROUND: Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. RESULTS: In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. CONCLUSIONS: This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

Transformed tissue of Dionaea muscipula J. Ellis as a source of biologically active phenolic compounds with bactericidal properties.

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, and Escherichia coli and up to 7% against Pseudomonas 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.

Stem Photosynthesis-A Key Element of Grass Pea (Lathyrus sativus L.) Acclimatisation to Salinity.

Grass pea (Lathyrus sativus) is a leguminous plant of outstanding tolerance to abiotic stress. The aim of the presented study was to describe the mechanism of grass pea (Lathyrus sativus L.) photosynthetic apparatus acclimatisation strategies to salinity stress. The seedlings were cultivated in a hydroponic system in media containing various concentrations of NaCl (0, 50, and 100 mM), imitating none, moderate, and severe salinity, respectively, for three weeks. In order to characterise the function and structure of the photosynthetic apparatus, Chl a fluorescence, gas exchange measurements, proteome analysis, and Fourier-transform infrared spectroscopy (FT-IR) analysis were done inter alia. Significant differences in the response of the leaf and stem photosynthetic apparatus to severe salt stress were observed. Leaves became the place of harmful ion (Na+) accumulation, and the efficiency of their carboxylation decreased sharply. In turn, in stems, the reconstruction of the photosynthetic apparatus (antenna and photosystem complexes) activated alternative electron transport pathways, leading to effective ATP synthesis, which is required for the efficient translocation of Na+ to leaves. These changes enabled efficient stem carboxylation and made them the main source of assimilates. The observed changes indicate the high plasticity of grass pea photosynthetic apparatus, providing an effective mechanism of tolerance to salinity stress.

Can Ceylon Leadwort (Plumbago zeylanica L.) Acclimate to Lead Toxicity?-Studies of Photosynthetic Apparatus Efficiency.

Ceylon leadwort (Plumbago zeylanica) is ornamental plant known for its pharmacological properties arising from the abundant production of various secondary metabolites. It often grows in lead polluted areas. The aim of presented study was to evaluate the survival strategy of P. zeylanica to lead toxicity via photosynthetic apparatus acclimatization. Shoots of P. zeylanica were cultivated on media with different Pb concentrations (0.0, 0.05, and 0.1 g Pb∙l-1). After a four-week culture, the efficiency of the photosynthetic apparatus of plants was evaluated by Chl a fluorescence measurement, photosynthetic pigment, and Lhcb1, PsbA, PsbO, and RuBisCo protein accumulation, antioxidant enzymes activity, and chloroplast ultrastructure observation. Plants from lower Pb concentration revealed no changes in photosynthetic pigments content and light-harvesting complex (LHCII) size, as well as no limitation on the donor side of Photosystem II Reaction Centre (PSII RC). However, the activity and content of antioxidant enzymes indicated a high risk of limitation on the acceptor side of Photosystem I. In turn, plants from 0.1 g Pb∙l-1 showed a significant decrease in pigments content, LHCII size, the amount of active PSII RC, oxygen-evolving complex activity, and significant remodeling of chloroplast ultrastructure indicated limitation of PSII RC donor side. Obtained results indicate that P. zeylanica plants acclimate to lead toxicity by Pb accumulation in roots and, depending on Pb concentration, by adjusting their photosynthetic apparatus via the activation of alternative (cyclic and pseudocyclic) electron transport pathways.

Elicitation-Based Method for Increasing the Production of Antioxidant and Bactericidal Phenolic Compounds in Dionaea muscipula J. Ellis Tissue.

The carnivorous plant Dionaea muscipula J. Ellis (Venus flytrap) is a widely known medical herb, capable of producing various phenolic compounds known for their strong antioxidant and antibacterial properties. In the pharmaceutical industry, Venus flytrap is grown in tissue cultures, as the natural population of D. muscipula is very limited. Here, we describe an improved method to increase the quantity and quality of phenolic compounds produced in D. muscipula. This is achieved by combining biotic elicitation (using Cronobacter sakazakii bacteria lysate) of D. muscipula cultured with rotary shaking (hydromechanical stress), which we describe here for the first time. The antibacterial activity and the antioxidant properties of the obtained compounds were studied on two antibiotic-resistant human pathogenic bacteria. The proposed plant culture conditions resulted in an increase in fresh weight, as well as a higher total phenolic content, in comparison to traditional tissue cultures on agar-solidified medium. With the use of high-performance liquid chromatography, we demonstrated that the described elicitation strategy leads to an increased synthesis of myricetin, caffeic acid, ellagic acid and plumbagin in D. muscipula tissue. We also found that a higher level of antioxidant activity, exhibited by the plant extract, corresponded with its higher phenylpropanoid content. The bactericidal activity of the extract against Staphylococcus aureus was dependent on the duration of plant culture under described elicitation conditions, whereas neither elicitation condition (duration or elicitor concentration) seemed relevant for the bactericidal activity of the extract towards Escherichia coli. This suggest that Gram-negative bacteria are less sensitive to compounds derived from Venus flytrap tissue.

Human erythrocytes under stress. Spectroscopic fingerprints of known oxidative mechanisms and beyond.

In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (H2O2) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than H2O2 and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of ß-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and H2O2 caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into ß-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.

Effect of silver nanoparticles foliar application on the nutritional properties of potato tubers.

The aim of presented study was to test nutritional properties of potato tubers and silver ions accumulation pattern after foliar application of silver nanoparticles (AgNPs) during potato vegetation. Potato plants were sprayed with different concentration of Ag nanoparticles (0.1, 1.0 and 10 mg·dm-3) synthesized with incorporation with sodium dodecyl sulphate (SDS) and sodium citrate as stabilizing agent. The lowest amounts of silver ions were transported to the tubers after spraying with AgNPs synthesized with SDS, rather than with citrate. Nevertheless silver ions accumulation in tubers was negligible. SDS method of synthesis was more favourable in terms of nutritional properties of potato tubers. The highest tested concentration of AgNPs_SDS had a favourable effect on a variety of macro- and micronutrients, ascorbic acid and soluble sugars. In turn, lower concentrations of AgNPs_SDS increased the content of phenolic compounds and free radical scavenging efficiency of tubers. These correlations were also confirmed by Principal Component Analysis.

Effect of Agitation and Temporary Immersion on Growth and Synthesis of Antibacterial Phenolic Compounds in Genus Drosera.

Sundews (Drosera sp.) are the source of biologically active secondary metabolites: phenolic acids, flavonoids, and 1,4-naphtoquinones. Because obtaining them from the natural environment is impossible (rare and endangered species), in this study modifications of traditional tissue cultures grown in solid medium (SM), such as agitated cultures (ACs) (cultures in liquid medium with rotary shaking) and temporary immersion bioreactors PlantformTM (TIB), were used for multiplication of four sundew species: Drosera peltata, Drosera indica, Drosera regia, and Drosera binata, with simultaneously effective synthesis of biologically active phenolic compounds. Each species cultivated on SM, AC, and TIB was tested for biomass accumulation, the content of total phenols and selected phenolic derivative concentrations (DAD-HPLC), the productivity on of phenolic compounds, as well as its antibacterial activity against two human pathogens: Staphylococcus aureus and Escherichia coli. The results showed that the type of culture should be selected for each species separately. Phytochemical analyses showed that the synthesis of secondary metabolites from the groups of phenolic acids, flavonoids, and 1,4-naphthoquinones can be increased by modifying the cultivation conditions. D. regia turned out to be the richest in phenolic compounds, including 1,4-naphtoquinones: plumbagin and ramentaceone. Extracts from D. indica and D. regia tissue showed strong antibacterial activity against both pathogens. It has also been shown that the growth conditions of sundews can modify the level of secondary metabolites, and thus, their biological activity.

Assessment of Shoot Priming Efficiency to Counteract Complex Metal Stress in Halotolerant Lobularia maritima.

The study investigated whether short-term priming supports plant defense against complex metal stress and multiple stress (metals and salinity) in halophyte Lobularia maritima (L.) Desv. Plants were pre-treated with ectoine (Ect), nitric oxide donor-sodium nitroprusside (SNP), or hydrogen sulfide donor-GYY4137 for 7 days, and were transferred onto medium containing a mixture of metal ions: Zn, Pb, and Cd. To test the effect of priming agents in multiple stress conditions, shoots were also subjected to low salinity (20 mM NaCl), applied alone, or combined with metals. Hydropriming was a control priming treatment. Stress impact was evaluated on a basis of growth parameters, whereas defense responses were on a basis of the detoxification activity of glutathione S-transferase (GST), radical scavenging activity, and accumulation of thiols and phenolic compounds. Exposure to metals reduced shoot biomass and height but had no impact on the formation of new shoots. Priming with nitric oxide annihilated the toxic effects of metals. It was related to a sharp increase in GST activity, glutathione accumulation, and boosted radical scavenging activity. In NO-treated shoots level of total phenolic compounds (TPC) and flavonoids remained unaffected, in contrast to other metal-treated shoots. Under combined metal stress and salinity, NO and H2S were capable of restoring or improving growth parameters, as they stimulated radical scavenging activity. Ect and H2S did not exert any effect on metal-treated shoots in comparison to hydropriming. The results revealed the stimulatory role of nitric oxide and low doses of NaCl in combating the toxic effects of complex metal stress in L. maritima. Both NO and NaCl interfered with thiol metabolism and antioxidant activity, whereas NaCl also contributed to the accumulation of phenolic compounds.

Performance of the Photosynthetic Apparatus under Glass with a Luminophore Modifying Red-To-Far-Red-Light Ratio-A Case Study.

The aim of this study was to examine the effect of the modified light spectrum of glass containing red luminophore on the performance of the photosynthetic apparatus of two types of lettuce cultivated in soil in a greenhouse. Butterhead and iceberg lettuce were cultivated in two types of greenhouses: (1) covered with transparent glass (control) and (2) covered with glass containing red luminophore (red). After 4 weeks of culture, structural and functional changes in the photosynthetic apparatus were examined. The presented study indicated that the red luminophore used changed the sunlight spectrum, providing an adequate blue:red light ratio, while decreasing the red:far-red radiation ratio. In such light conditions, changes in the efficiency parameters of the photosynthetic apparatus, modifications in the chloroplast ultrastructure, and altered proportions of structural proteins forming the photosynthetic apparatus were observed. These changes led to a decrease of CO2 carboxylation efficiency in both examined lettuce types.

The Adjustment Strategy of Venus Flytrap Photosynthetic Apparatus to UV-A Radiation.

The objective of this study was to investigate the response of the photosynthetic apparatus of the Venus flytrap (Dionaea muscipula J. Ellis) to UV-A radiation stress as well as the role of selected secondary metabolites in this process. Plants were subjected to 24 h UV-A treatment. Subsequently, chl a fluorescence and gas exchange were measured in living plants. On the collected material, analyses of the photosynthetic pigments and photosynthetic apparatus proteins content, as well as the contents and activity of selected antioxidants, were performed. Measurements and analyses were carried out immediately after the stress treatment (UV plants) and another 24 h after the termination of UV-A exposure (recovery plants). UV plants showed no changes in the structure and function of their photosynthetic apparatus and increased contents and activities of some antioxidants, which led to efficient CO2 carboxylation, while, in recovery plants, a disruption of electron flow was observed, resulting in lower photosynthesis efficiency. Our results revealed that D. muscipula plants underwent two phases of adjustment to UV-A radiation. The first was a regulatory phase related to the exploitation of available mechanisms to prevent the over-reduction of PSII RC. In addition, UV plants increased the accumulation of plumbagin as a potential component of a protective mechanism against the disruption of redox homeostasis. The second was an acclimatization phase initiated after the running down of the regulatory process and decrease in photosynthesis efficiency.

Is a blue-red light a good elicitor of phenolic compounds in the family Droseraceae? A comparative study.

Plants from the family Droseraceae, especially Drosera sp. and Dionaea sp., are naturally rich in phenolic derivatives such as plumbagin, among others. Plumbagin is known both for its pharmacological significance and its protective properties against light stress. Light stress - high light intensity or/and light spectral composition - activates plants' response mechanisms including, among others, hormonal (salicylic acid, jasmonic acid) pathways and secondary metabolite (phenolic compounds, proline) pathways. Short-wavelength radiation, due to its high energy, will induce the synthesis of protective secondary metabolites, including those with pharmaceutical properties. The aim of the study was to describe and compare acclimation strategies of Drosera peltata and Dionaea muscipula to blue-red light in the context of phenolic compound accumulation, and salicylic acid, jasmonic acid and proline synthesis. For the first time, differences in the responses of D. muscipula and D. peltata to blue-red light (in the ratio 6:1) were established. In Dionaea sp., it was associated with the use of redox equivalents (in particular, plastoquinone pool) for the synthesis of primary metabolites used in the process of growth and development. In Drosera sp., a rapid adjustment of redox state led to the synthesis of secondary metabolites, constituting a reservoir of carbon skeletons and allowing for a quick defence response to stress factors. In both species, blue-red light did not induce the jasmonic acid pathway. However, the salicylic acid pathway was induced as an alternative to the phenolic compound synthesis pathway. Nevertheless, the applied blue-red light was not an effective elicitor of phenolic compounds in the plants examined.

The acclimatization strategies of kidney vetch (Anthyllis vulneraria L.) to Pb toxicity.

Kidney vetch (Anthyllis vulneraria L.) is a well-known Zn hyperaccumulator. Zn often occurs with Pb in one ore; thus, plants inhabiting waste dumps are exposed not only to Zn but also to Pb toxicity. While the response of kidney vetch to Zn toxicity is relatively well known, the Pb survival strategy of Anthyllis vulneraria has not been the subject of investigations. The aim of presented research was to determine the survival strategy of kidney vetch exposed to high lead concentrations. Shoot explants of a calamine kidney vetch ecotype were placed on agar media containing 0.0, 0.5, 1.0, and 1.5 mM Pb. Morphological, physiological, and biochemical responses, in particular photosynthetic apparatus of plantlets, were examined. The most pronounced changes were observed in plants grown on media supplemented with 1.5 mM Pb after 8 weeks of culture. Increased dry weight and high lead accumulation were observed in roots. Similarly, in shoots, increased dry weight and a decreased number of newly formed shoots were recorded. The accumulation of lead was many times lower in shoots than in roots. In leaf cells' ultra-structure, looser arrangement of chloroplast thylakoid grana was observed. Despite the decrease in chlorophyll a and carotenoid content, the photosynthetic apparatus remained efficient due to the lack of photoinhibition and increased electron transport rate beyond photosystem II (PSII). For the first time, an acclimatization mechanism based on maintaining the high efficiency of photosynthetic apparatus resulting from increasing of electron transport rate was described.