Catmint (Nepeta nuda L.) Phylogenetics and Metabolic Responses in Variable Growth Conditions.

Nepeta nuda (catmint; Lamiaceae) is a perennial medicinal plant with a wide geographic distribution in Europe and Asia. This study first characterized the taxonomic position of N. nuda using DNA barcoding technology. Since medicinal plants are rich in secondary metabolites contributing to their adaptive immune response, we explored the N. nuda metabolic adjustment operating under variable environments. Through comparative analysis of wild-grown and in vitro cultivated plants, we assessed the change in phenolic and iridoid compounds, and the associated immune activities. The wild-grown plants from different Bulgarian locations contained variable amounts of phenolic compounds manifested by a general increase in flowers, as compared to leaves, while a strong reduction was observed in the in vitro plants. A similar trend was noted for the antioxidant and anti-herpesvirus activity of the extracts. The antimicrobial potential, however, was very similar, regardless the growth conditions. Analysis of the N. nuda extracts led to identification of 63 compounds including phenolic acids and derivatives, flavonoids, and iridoids. Quantification of the content of 21 target compounds indicated their general reduction in the extracts from in vitro plants, and only the ferulic acid (FA) was specifically increased. Cultivation of in vitro plants under different light quality and intensity indicated that these variable light conditions altered the content of bioactive compounds, such as aesculin, FA, rosmarinic acid, cirsimaritin, naringenin, rutin, isoquercetin, epideoxyloganic acid, chlorogenic acid. Thus, this study generated novel information on the regulation of N. nuda productivity using light and other cultivation conditions, which could be exploited for biotechnological purposes.

In Vitro Multiplication and NMR Fingerprinting of Rare Veronica caucasica M. Bieb.

Micropropagation of rare Veronica caucasica M. Bieb. was achieved by successful in vitro cultivation of mono-nodal segments on MS medium supplemented with 1.0 mg L-1 6-benzylaminopurine (BA) and then transferring the regenerated plants on hormone free basal MS medium for root development. In vitro multiplicated plants were successively acclimated in a growth chamber and a greenhouse with 92% survival. The number of plastid pigments and the total phenolics content in in vitro cultivated and ex vitro adapted plants were unchanged, and no accumulation of reactive oxygen species (ROS) was detected by staining with 3-3'-diaminobenzidine (DAB) and 2',7'-dichlorofluorescein diacetate (DCF-DA). Nuclear Magnetic Resonance (NMR) fingerprinting allowed for the identification of the major alterations in metabolome of V. caucasica plants during the process of ex situ conservation. Iridoid glucosides such as verproside, aucubin and catalpol were characteristic for in vitro cultivated plants, while in ex vitro acclimated plants phenolic acid-protocatechuic acid and caffeic acid appeared dominant. The successful initiation of in vitro and ex vitro cultures is an alternative biotechnological approach for the preservation of V. caucasica and would allow for further studies of the biosynthetic potential of the species and the selection of lines with a high content of pharmaceutically valuable molecules and nutraceuticals.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

OBJECTIVES: To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. RESULTS: Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. CONCLUSION: Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Changes in the functional characteristics of tumor and normal cells after treatment with extracts of white dead-nettle.

Lamium album L. is a perennial herb widely used in folk medicine. It possesses a wide spectrum of therapeutic activities (anti-inflammatory, astringent, antiseptic, antibiotic, antispasmodic, antioxidant and anti-proliferative). Preservation of medicinal plant could be done by in vitro propagation to avoid depletion from their natural habitat. It is important to know whether extracts from L. album plants grown in vitro possess similar properties as extracts from plants grown in vivo. For these reasons, it is important to examine changes in the composition of secondary metabolites during in vitro cultivation of the plant and how they affect the biological activity. We used A549 human cancer cell line and normal kidney epithelial cells MDCKII (Madin-Darby canine kidney cells II) as controls in assessing the anti-cancer effect of plant extracts. To elucidate changes in some key functional characteristics, adhesion test, MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER), immunofluorescence staining and trypan blue exclusion test were performed. Methanol and chloroform extracts of in vivo and in vitro propagated plants affected differently cancerous and non-cancerous cells. The most pronounced differences were observed in the morphological analysis and in the cell adhesive properties. We also detected suppressed epithelial transmembrane electrical resistance of MDCK II cells, by treatment with plant extracts, compared to non-treated MDCK II cells. A549 cells did not polarize under the same conditions. Altered organization of actin filaments in both cell types were noticed suggesting that extracts from L. album L. change TER and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II cells.

Influence of cryopreservation on the antioxidative activity of in vitro cultivated Hypericum species.

Antioxidative activity of two in vitro cultivated Hypericum species - H. rumeliacum Boiss. and H. tetrapterum Fr. - was estimated after cryopreservation. Both species were successfully regenerated after a cryopreservation procedure performed by the vitrification method. H. tetrapterum did not manifest any significant oxidative stress-induced changes caused by low-temperature treatment. Conversely, a decrease in green pigments' content of H. rumeliacum was measured, particularly pronounced in chlorophyll b, which was accompanied by an increase of carotenoids in the regenerated plants. A strong increase of malone dialdehyde and H2O2 levels in H. rumeliacum tissues was detected. Superoxide dismutase activity was enhanced by 170%, as well as the catalase activity, which was 220% above the control. The same trend was observed in H. tetrapterum, although less pronounced - 143% increase of superoxide dismutase and 112% of catalase. Cryopreservation did not influence the phenol content in the examined plants, but it led to an increase of flavonoid content, especially in H. tetrapterum, by 237%. Total antioxidant activity in regenerated H. tetrapterum varied around the control level, but it was increased in H. rumeliacum. The free proline content in H. tetrapterum remained almost unaffected after freezing, as opposed to H. rumeliacum, where a strong increase of proline content (208% above the control) occurred. An electrolyte leakage from the cells of H. rumeliacum regenerated after cryopreservation was also registered, albeit not significant.

Antimicrobial activity of extracts from in vivo and in vitro propagated Lamium album L. plants.

The antimicrobial activity of 18 different extracts from in vivo and in vitro grown L. album L. plants was evaluated against clinical bacteria and yeasts using the well diffusion method. All the used extracts demonstrated antibacterial activity, whereas only the water extracts from leaves (in vivo) possessed antifungal activity against Candida albicans NBIMCC 72 and Candida glabrata NBIMCC 8673 (14 and 20 mm diameter of inhibition zones and MIC 10 mg/ml, respectively). The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those from in vivo plants, while the opposite tendency was observed for the chloroform extracts. All tested flower extracts possessed antimicrobial activity. The chloroform extract from in vivo flowers demonstrated the highest activity against E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339 and P. aeruginosa NBIMCC 3700 (22 mm, 13 mm, 11 mm, 23 mm zone diameter of inhibition and MIC 0.313 mg/ml, respectively). The water extracts from leaves (both in vivo and in vitro) possessed higher antibacterial activity than extract from flowers. The obtained results showed that both in vivo and in vitro propagated L. album L. could be used as a source of antibacterial substances.

Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii.

BACKGROUND AND AIMS: Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. METHODS: Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. KEY RESULTS: MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. CONCLUSIONS: In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways.

Oxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature treatment.

Extreme low temperatures cause plants multiple stresses, among which oxidative stress is presumed to be the major component affecting the resultant recovery rate. Plants of Hypericum perforatum L., which are known especially for the photodynamic activities of hypericins capable of producing reactive oxygen species under exposure to visible light, were observed to display a substantial increase and persistence in active oxygen production at least two months after recovery from cryogenic treatment. In an effort to uncover the causative mechanism, the individual contributions of wounding during explant isolation, dehydration and cold were examined by means of antioxidant profiling. The investigation revealed activation of genes coding for enzymatic antioxidant catalase and superoxide dismutase at both the transcript and protein levels. Interestingly, plants responded more to wounding than to either low-temperature associated stressor, presumably due to tissue damage. Furthermore, superoxide dismutase zymograms showed the Cu/Zn isoforms as the most responsive, directing the ROS production particularly to chloroplasts. Transmission electron microscopy revealed chloroplasts as damaged structures with substantial thylakoid ruptures.

Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot tips.

Hypericum perforatum L. in vitro cultured shoot tips were characterised at the physiological, biochemical and molecular levels following recovery from cryogenic treatment using the plant vitrification solutions PVS2 and PVS3. This comparative study revealed an increase in recovery and regrowth of explants cryoprotected with PVS3. Among the physiological markers only lipid peroxidation in the regenerants treated with PVS2 significantly increased indicating membrane damage. Genotype-specific interactions were found in most characteristics studied, with some variation detected within control and cryopreserved samples. Analyses of metabolite biosynthesis and genetic stability showed no significant differences in hypericin content, RAPD and minisatellite amplification profiles between PVS2- and PVS3-treated explants. This study demonstrates and discusses the criteria selective for PVS3 to improve the cryopreservation of H. perforatum L.

Involvement of ethylene and nitric oxide in cell death in mastoparan-treated unicellular alga Chlamydomonas reinhardtii.

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)-induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene- and NO-interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP-induced cell death. By employing a carbon dioxide laser-based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)-based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP-induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP-induced cell death.

Cadmium toxicity in cultured tomato cells--role of ethylene, proteases and oxidative stress in cell death signaling.

Our aim was to investigate the ability of cadmium to induce programmed cell death in tomato suspension cells and to determine the involvement of proteolysis, oxidative stress and ethylene. Tomato suspension cells were exposed to treatments with CdSO(4) and cell death was calculated after fluorescein diacetate staining of the living cells. Ethylene was measured in a flow-through system using a laser-driven photo acoustic detector; hydrogen peroxide was determined by chemiluminescence in a ferricyanide-catalysed oxidation of luminol. We have demonstrated that cadmium induces cell death in tomato suspension cells involving caspase-like proteases, indicating that programmed cell death took place. Using range of inhibitors, we found that cysteine and serine peptidases, oxidative stress, calcium and ethylene are players in the cadmium-induced cell death signaling. Cadmium-induced cell death in tomato suspension cells exhibits morphological and biochemical similarities to plant hypersensitive response and to cadmium effects in animal systems.

Signal transduction events in aluminum-induced cell death in tomato suspension cells.

In this study, some of the signal transduction events involved in AlCl(3)-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 microM AlCl(3) showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation. Cell death was effectively inhibited by protease and human caspase inhibitors indicating a cell death execution mechanism with similarities to animal apoptosis. Cell death was suppressed by application of antoxidants and by inhibitors of phospholipase C (PLC), phospholipase D (PLD) and ethylene signalling pathways. The results suggest that low concentrations of heavy metal ions stimulate both PLC and PLD signalling pathways leading to the production of reactive oxygen species (ROS) and subsequent cell death executed by caspase-like proteases.