Marzena Wielanek

Glucotropaeolin and myrosinase production in hairy root cultures of Tropaeolum majus

We obtained callus, cell suspension and hairy root cultures of Tropaeolum majus and we demonstrated their ability to produce glucotropaeolin and myrosinase. In hairy roots glucotropaeolin content and myrosinase activity were higher in comparison with callus, cell suspension and leaves of intact plants. In hairy root cultures the highest glucotropaeolin contents were detected on the 9th day of culture. In relation to dry weight glucotropaeolin production by hairy roots was stimulated markedly by cystein and less by phenylalanine and methyl jasmonate. Cystein inhibited biomass growth so in relation to culture volume stimulating effect of cystein on glucotropaeolin yield declined. Myrosinase activity was stimulated by methyl jasmonate. The ratio of myrosinase activity to glucotropaeolin content in cultures with methyl jasmonate was much higher than in cultures with cystein.

Involvement of salicylic acid, glutathione and protein S-thiolation in plant cell death-mediated defence response of Mesembryanthemum crystallinum against Botrytis cinerea

The response of Mesembryanthemum crystallinum plants performing C3 photosynthesis and crassulacean acid metabolism (CAM) to the non-host necrotrophic pathogen Botrytis cinerea was analyzed at the local and systemic levels. The induction of programmed cell death, lignin and callose deposition, changes in salicylic acid, glutathione and cysteinylglycine pools as well as the content of thiolated proteins were studied. The infected C3 and CAM plants exhibited hypersensitive-like defence response, however fluorescence staining with acridine orange and ethidium bromide revealed programmed cell death events in C3 plants only. The local immune response was not related to callose and lignin deposition. In the infected plants, salicylic acid, glutathione and cysteinylglycine, the first product of glutathione catabolism, as well as protein S-thiolation, predominantly S-glutathionylation, contributed to local defence at sites of inoculation. They (except protein thiolation) were also active in the establishment of systemic acclimation response monitored in the non-treated upper leaves. The extent to which they were involved in the local and systemic responses induced by B. cinerea differed in C3 and CAM plants. The accumulation of free salicylic acid, both in treated and upper leaves of the infected plants, was much more pronounced in CAM plants. The results have been discussed with respect to redox regulations in defence against necrotrophic pathogens and to stress acclimation.

A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus L.

Leonurus sibiricus L. is a traditional medicinal plant which occurs in southern Siberia, China, Korea, Japan, and Vietnam. The plant shows several pharmacological effects, but the most interesting is its anti-cancer activity. The aim of our study was to examine the induction of apoptosis in malignant glioma cells, the most aggressive primary brain tumors of the central nervous system, following treatment with transformed root (TR) or non-transformed root (NR) L. sibiricus extracts. Both the NR and TR extracts were found to have cytotoxic activity in the glioma primary cells. The human glioblastoma cell lines obtained from patients were confirmed to be tumorogenic by the following three markers: D10S1709, D10S1172, and D22S283. HPLC and MS analysis revealed the presence of polyphenolic compounds (chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid, ellagic acid, and verbascoside) in both sets of root extracts. In summary, our findings demonstrate that treatment of the glioma cells with NR and TR extracts resulted (a) in significant cell growth inhibition, (b) S- and G2/M-phase cell cycle arrest, and (c) apoptosis in a dose-dependent fashion by changing Bax/Bcl-2 ratio (about 4-fold increase) and p53 (5-fold increase) activation. These findings indicate that NR and TR extracts exhibit anti-cancer activity through the regulation of genes involved in apoptosis. This is the first report to demonstrate the cytotoxic effect of polyphenolic extracts from L. sibiricus roots against glioma cells, but further studies are required to understand the complete mechanism of its apoptosic activity.

The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

Alleviation of nickel toxicity in wheat (Triticum aestivum L.) seedlings by selenium supplementation

Abstract Hydroponically grown wheat seedlings were treated with 50 μM N i and/or 15 μM Se. After a 7-day culture period, their growth parameters, N i, Se, F e, and M g contents, electrolyte leakage, photosynthetic pigment concentrations, and photochemical activity of photosystem II were determined. Exposure of wheat seedlings to N i alone resulted in reduction in the total shoot and root lengths, by 22% and 50%, respectively. Addition of Se to the N i-containing medium significantly improved the growth of these organs, compared to the seedlings subjected to N i alone. Application of Se decreased the accumulation of N i in shoots and roots and partially alleviated the N i-induced decrease in F e and M g concentations in shoots. Electrolyte leakage increased in response to N i stress, but in shoots it was diminished by Se supplementation. Exposure to N i led to a decrease in chlorophyll a and b contents and enhancement of chlorophyll a/b ratio, but did not influence the concentration of carotenoids. Enrichment of the N i-containing medium with Se significantly increased chlorophyll b content, compared to the seedlings treated with N i alone. Photochemical activity, estimated in terms of the maximum quantum yield of photosystem II , decreased in response to N i treatment but was significantly improved by simultaneous addition of Se. Results of our study suggest that alleviation of N i toxicity in wheat seedlings by Se supplementation may be related to limitation of N i uptake.

An automatic segmentation method for scanned images of wheat root systems with dark discolourations

An automatic segmentation method for scanned images of wheat root systems with dark discolourations The analysis of plant root system images plays an important role in the diagnosis of plant health state, the detection of possible diseases and growth distortions. This paper describes an initial stage of automatic analysis—the segmentation method for scanned images of Ni-treated wheat roots from hydroponic culture. The main roots of a wheat fibrous system are placed separately in the scanner view area on a high chroma background (blue or red). The first stage of the method includes the transformation of a scanned RGB image into the HCI (Hue-Chroma-Intensity) colour space and then local thresholding of the chroma component to extract a binary root image. Possible chromatic discolourations, different from background colour, are added to the roots from blue or red chroma subcomponent images after thresholding. At the second stage, dark discolourations are extracted by local fuzzy c-means clustering of an HCI intensity image within the binary root mask. Fuzzy clustering is applied in local windows around the series of sample points on roots medial axes (skeleton). The performance of the proposed method is compared with hand-labelled segmentation for a series of several root systems.

Over-Expression of AtPAP1 Transcriptional Factor Enhances Phenolic Acid Production in Transgenic Roots of Leonurus sibiricus L. and Their Biological Activities

This study examines the production of five phenolic acids (chlorogenic acid, neochlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) following over-expression of AtPAP1 transcription factor by four transgenic root clones of Leonurus sibiricus after Agrobacterium rhizogenes transformation. The AtPAP1 expression level was estimated by quantitative real-time PCR. High levels of phenolic acids were found in the transgenic roots of L. sibiricus and were determined by high-performance liquid chromatography–mass spectrometry analysis. Additionally, transgenic roots showed antimicrobial potential and cytotoxic activity on glioma cells in IV grade. Our results suggest that L. sibiricus transformed roots with AtPAP1 gene over-expression may represent a potential source of phenolic acids.

A Method for Thiarubrine Canals Extraction in Optical Coherence Tomography Images of Schkuhria Pinnata Roots

Abstract This paper presents a method of automatic recognition of thiarubrine canals in images obtained with Optical Coherence Tomography technique. The plant material was the Ri-transformed root culture of South American herb Schkuhria pinnata. The series of highresolution OCT B-scans for the study were collected using custom made experimental system operating light of 800 nm central wavelength. The method reduces significant artefacts and uses region growing approach adapted to specific features of OCT images. Results of the identification have been compared with data obtained by specialist for selected B-scans. The algorithm accuracy was also verified using a simple numeric phantom.

Phenolic profiles in apple leaves and the efficacy of selected phenols against fire blight (Erwinia amylovora)

The content and type of phenolic compounds in apple leaves as potential markers of resistance to fire blight were analysed. The amounts of phenolic acids and flavonoids were determined before and after E. amylovora inoculation of leaves of two cultivars: ‘Enterprise’ (highly resistant) and ‘Idared’ (highly susceptible). The basic levels of phenolics in both cultivars was similar but, following the inoculation, in the resistant one faster and more distinguishable changes were observed. The difference between the cultivars was related to the content of the compounds and the rate of release of free phenols from their glucosides. Regarding age dependency, the levels of eight out of 15 phenolics was significantly higher in young leaves of ‘Idared” than in ‘Enterprise’. In the older leaves the differences were limited to four compounds. The amount of salicylic acid in ‘Idared’ was lower than in ‘Enterprise’. In ‘Idared’ accumulation of salicylate after infection was better pronounced than in ‘Enterprise’. Higher levels of naringenin glucosides, 4-hydroxbenzoic acid and gentisic acid were found in ‘Enterprise’. The activity of 13 phenolics tested in vitro against the pathogen showed that gallic acid, phloroglucinol, hydroquinone and phloretin, suppressed its growth. The aqueous solutions of gallic acid, phloroglucinol and hydroquinone also significantly limited the development of disease on pear fruitlet slices but only hydroquinone maintained its protective activity for longer time. It also showed very high efficacy in preventing disease spread on apple shoots. The study adds novel information on the contribution of specific phenolics to apple resistance to fire blight.