Živko Jovanović

Characterization of oxidative and antioxidative events during dehydration and rehydration of resurrection plant Ramonda nathaliae

In order to investigate changes of oxidative status in relation to the activity of the various protective mechanisms in resurrection plant Ramonda nathaliae, we have analysed time and relative water content (RWC) related changes in lipid peroxidation and ion leakage, hydrogen peroxide accumulation, changes of pigment content and antioxidative enzyme activity, together with expression of dehydrins. The results indicate that enhanced oxidative status during dehydration, not previously reported for resurrection plants, could play an active role in inducing the desiccation adaptive response in R. nathaliae. A critical phase is shown to exist during dehydration (in the range of RWC between 50 and 70%) during which a significant increase in hydrogen peroxide accumulation, lipid peroxidation and ion leakage, accompanied by a general decline in antioxidative enzyme activity, takes place. This phase is designated as a transition characterized by change in the type of stress response. The initial response, relying mainly on the enzymatic antioxidative system, is suspended but more effective, desiccation specific protective mechanisms, such as expression of dehydrins, are then switched on. The expression of dehydrins in R. nathaliae could be inducible as well as constitutive. In order to cope with the oxidative stress associated with rapid rewatering, R. nathaliae reactivated antioxidative enzymes. We propose that controlled elevation of reactive oxygen species, such as hydrogen peroxide, could be an important mechanism enabling resurrection plants to sense dehydration and to trigger an adaptive programme at an appropriate stage during the dehydration/rehydration cycle.

Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum).

Metallothionein type 3 (MT3) expression has previously been detected in leaves, fruits, and developing somatic embryos in different plant species. However, specific tissular and cellular localization of MT3 transcripts have remained unidentified. In this study, in situ RNA-RNA analysis revealed buckwheat metallothionein type 3 (FeMT3) transcript localization in vascular elements, mesophyll and guard cells of leaves, vascular tissue of roots and throughout the whole embryo. Changes in FeMT3 mRNA levels in response to drought and oxidative stress, as well as ROS scavenging abilities of the FeMT3 protein in yeast were also detected, indicating possible involvement of FeMT3 in stress defense and ROS related cellular processes.

Water deficit down-regulates miR398 and miR408 in pea (Pisum sativum L.).

MicroRNAs (miRNAs), recently recognized as important regulator of gene expression at posttranscriptional level, have been found to be involved in plant stress responses. The observation that some miRNAs are up- or down regulated by stress implies that they could play vital roles in plant resistance to abiotic and biotic stress. We investigated the effect of water stress treatment during 10 days on expression of conserved miRNAs-miR398a/b and miR408 in pea plants. This time frame reflects the changes as close as possible to the changes where water stress causes visible effects under field condition. It was observed that dehydration strongly down regulates the expression of both miR398a/b and miR408 in pea roots and shoots. The down-regulation of miR398a/b and the up-regulation of potential target genes - copper superoxide dismutase, CSD1, highlight the involvement of this miRNA in pea stress response. To the contrary, the mRNA level of cytochrome c oxidase subunit 5 (COX5b) did not change in roots and shoots of water-stressed plants, compared to control (well) hydrated plants. This suggests that COX5b is not the target of miR398, or that its expression is regulated by some other mechanism. P1B-ATPase expression increased during water deficit only in the shoots of pea; in the roots there were no changes in expression. Our results help to understand the possible role of investigated miRNAs and their contribution to pea capacity to cope with water deficit.

Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids

Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a–h) and eight of their diacylhydrazine precursors (6a–h) were synthesized, characterized using spectroscopic methods and examined by scavenging of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity, moderate to good H2O2 scavenging properties and strong ferric ion reducing capacity. Further in vitro evaluation of the antioxidant properties of the most active compounds demonstrated their protective effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress. Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison with a control sample and did not affect the superoxide dismutase activity.

Resurrection plants of the genus Ramonda: prospective survival strategies – unlock further capacity of adaptation, or embark on the path of evolution?

Paleoendemic species of the monophyletic genus Ramonda (R. myconi, R. serbica and R.~nathaliae) are the remnants of the Tertiary tropical and subtropical flora in Europe. They are the rare resurrection plants of Northern Hemisphere temperate zone. Ramonda serbica and R. nathaliae are chorologically differentiated in the Balkan Peninsula and occupy similar habitats in calcareous, northward slopes in canyons and mountainsides. They remain well-hydrated during spring, late autumn and even in winter. In summer and early autumn when plants are subjected to drought and thermal stress, their desiccation tolerance comes into operation and they fall into anabiosis. Investigations revealed the permanent presence of ubiquitine and its conjugates, high amounts of oxalic acid and proline. Both species are homoiochlorophyllous. It enables them to rapidly resume photosynthesis upon rehydration, but also makes them susceptible to reactive oxygen species formation. Dehydration induces activation of antioxidative enzymes (ascorbate peroxidase, glutathione reductase, polyphenol oxidase), increase in amounts of AsA and GSH, phenolic acids, dehydrins, sucrose, and inorganic ions. Plasma membranes, characterized by high amount of cholesterol, are subjected to decrease in membrane fluidity mostly on account of increased level of lipid saturation. Cytogenetic analysis revealed that R. nathaliae is a diploid (2n = 48) and probably evolutionary older species, while R. serbica is a hexaploid (2n = 144). Two species live together in only two localities forming hybrid individuals (2n = 96). Polyploidization is the major evolutionary mechanism in the genus Ramonda that together with hybridization ability indicates that these relict species which have preserved an ancient survival strategy are not the evolutionary “dead end.”The species of the genus Ramonda are promising sources of data important for understanding the complex strategy of resurrection plants’ survival, appraised through a prism of their evolutionary and adaptive potential for multiple environmental stresses.

Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities

ABSTRACT To date little has been done on identification of major phenolic compounds responsible for anticancer and antioxidant properties of pea (Pisum sativum L.) seed coat extracts. In the present study, phenolic profile of the seed coat extracts from 10 differently colored European varieties has been determined using ultrahigh-performance liquid chromatography–linear trap quadrupole orbitrap mass spectrometer technique. Extracts of dark colored varieties with high total phenolic content (up to 46.56 mg GAE/g) exhibited strong antioxidant activities (measured by 2,2-diphenyl-1-picrylhydrazyl or DPPH assay, and ferric ion reducing and ferrous ion chelating capacity assays) which could be attributed to presence of gallic acid, epigallocatechin, naringenin, and apigenin. The aqueous extracts of dark colored varieties exert concentration-dependent cytotoxic effects on all tested malignant cell lines (human colon adenocarcinoma LS174, human breast carcinoma MDA-MB-453, human lung carcinoma A594, and myelogenous leukemia K562). Correlation analysis revealed that intensities of cytotoxic activity of the extracts strongly correlated with contents of epigallocatechin and luteolin. Cell cycle analysis on LS174 cells in the presence of caspase-3 inhibitor points out that extracts may activate other cell death modalities besides caspase-3-dependent apoptosis. The study provides evidence that seed coat extracts of dark colored pea varieties might be used as potential cancer-chemopreventive and complementary agents in cancer therapy.

Integrating archaeobotany, paleogenetics and historical linguistics may cast more light onto crop domestication: the case of pea (Pisum sativum)

This short communication intends to discuss various models and ideas about the very beginnings of plant domestication in the Old World with a specific emphasis on pea (Pisum sativum L.) as one of the most significant crops in the human civilization. It presents a selection of some recent achievements and challenges in studying the earliest days of this important grain legume species by balanced contributions from archaeobotany, paleogenetics and historical linguistics. It will hopefully stimulate a fruitful interdisciplinary discussion and provide a basis for new experimental approaches.