Veneta Kapchina-Toteva

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 CdSO4 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.

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.

Role of phenylurea cytokinin CPPU in apical dominance release in in vitro cultured Rosa hybrida L.

A bstractThe effect of purine (BA) and phenylurea (CPPU) cytokinins on apical dominance release in in vitro cultured Rosa hybrida L., cv. Madelon and Motrea was evaluated. Cv. Madelon shows stronger natural apical growth and fewer branches than cv. Motrea in vivo and in vitro. We examined the effects under three conditions, without the addition of the auxin IBA, in the presence of IBA, and in material pretreated with a pulse of IBA. Results were scored weekly for 4 weeks. BA and CPPU stimulated axillary bud break, and higher numbers of open buds were recorded in the presence of CPPU. When CPPU cytokinin was added to culture medium, physiologic features such as bud sprouting and shoot fresh and dry weight were enhanced. CPPU was also highly efficient for overcoming IBA inhibition of bud outgrowth. Different cultivar responses were observed.

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.

Involvement of ethylene, oxidative stress and lipid-derived signals in cadmium-induced programmed cell death in tomato suspension cells

Backgraund Extensive research is ongoing looking for the characterisation of programmed cell death (PCD) in plants involving pathogen attack, chemical elicitation and abiotic inducers, but there are still limited reports on the role of heavy metals in PCD induction and little is known about cadmium-triggered signal transduction in plant systems. Contamination of biosphere with heavy metals has hazardous effect on agricultural crops and human health. In animal models, cadmium intoxication occurs through apoptosis appearing by apoptotic phenotype and an oxidative stress is involved in the mechanism of Cd action. The goal of this present work was to investigate if programmed cell death occurs in cadmium-treated tomato suspension cells; to identify some of the biochemical processes contributing to the signal transduction pathway(s) involved in cadmium toxicity; to investigate the role of oxidative stress (hydrogen peroxide), ethylene and lipid-derived signals and to look for similarities between cadmiumand camptothecin-induced cell death.

Adaptive changes in photosynthetic performance and secondary metabolites during white dead nettle micropropagation.

The white dead nettle, Lamium album L., is an herb that has been successfully cultivated under in vitro conditions. The L. album micropropagation system offers a combination of factors (light intensity, temperature, carbon dioxide (CO2) level, humidity) that are limiting for plant growth and bioactive capacity. To get a better understanding of the mechanism of plant acclimation towards environmental changes, we performed a comparative investigation on primary and secondary metabolism in fully expanded L. album leaves during the consecutive growth in in situ, in vitro, and ex vitro conditions. Although the genetic identity was not affected, structural and physiological deviations were observed, and the level of bioactive compounds was modified. During in vitro cultivation, the L. album leaves became thinner with unaffected overall leaf organization, but with a reduced number of palisade mesophyll layers. Structural deviation of the thylakoid membrane system was detected. In addition, the photosystem 2 (PS2) electron transport was retarded, and the plants were more vulnerable to light damage as indicated by the decreased photoprotection ability estimated by fluorescence parameters. The related CO2 assimilation and transpiration rates were subsequently reduced, as were the content of essential oils and phenolics. Transfer of the plants ex vitro did not increase the number of palisade numbers, but the chloroplast structure and PS2 functionality were recovered. Strikingly, the rates of CO2 assimilation and transpiration were increased compared to in situ control plants. While the phenolics content reached normal levels during ex vitro growth, the essential oils remained low. Overall, our study broadens the understanding about the nature of plant responses towards environmental conditions.

Effect of Cytokinins and Cytokinin Antagonists on in vitro Cultured Gypsophila paniculata L.

This study deals with the effects of two cytokinins [kinetin (Kin) and N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)] and cytokinin antagonists [2-chloro-4-cyclobutyl-amino-6-ethylamino-1,3,5-triazine (ACK1) and N-(4-pyridyl)-O-(4-chlorophenyl)carbamate (ACK2)] in concentration of 1 μM on in vitro cultured Gypsophila. The application of Kin and CPPU stimulated bud opening and increased fresh and dry masses. Cytokinin antagonists reduced the number of sprouted buds and bud fresh and dry masses. In plants treated with CPPU the chloroplasts possessed well developed membrane system, which covered almost the entire chloroplasts volume. In ACK2 treated plants, the plastid apparatus in each cell was represented by two types of chloroplast in which the inner membrane system was differently organized. Cell wall adjacent chloroplasts possessed structure similar to the controls. In inner located chloroplasts part of thylakoids were semi-concentrically arranged and partially destructed.

Effect of N 6 -benzyladenine and indole-3-butyric acid on photosynthetic apparatus of Orthosiphon stamineus plants grown in vitro

The leaf structure and chloroplast ultrastructure of kidney tea (Orthosiphon stamineus Benth.) was studied in in vitro culture on standard MS medium supplemented with or without plant growth regulators (PGRs). The cytokinin N6-benzyladenine (BA) negatively affected the structure of the palisade parenchyma and chloroplast ultrastructure and increased the stomatal frequency of the adaxial epidermis. The auxin indole-3-butyric acid (IBA) did not modify the morphology of regenerated leaf tissues as well as the chloroplast ultrastructure. The effect of both PGRs applied in combination was manifested in well-differentiated mesophyll parenchyma, typical chloroplast ultrastructure and increased stomatal frequency on both leaf surfaces. This protocol can be suggested for further ex vitro propagation.

Anticytokinin Effect on Apical Dominance Release in in vitro Cultured Rosa Hybrida L.

Effects of two cytokinin antagonists - 2-chloro-4-cyclobutyl-amino-6-ethylamino-1,3,5-triazine and N-(4-pyridyl)-O-(4-chlorophenyl)carbamate - on bud break and outgrowth in single nodes from two Rosa hybrida cultivars differing in their apical dominance were studied. The compounds were applied at three different concentrations separately or in combination with benzyladenine. Cytokinin antagonists reduced the number of the sprouted buds in both cultivars at different extent. Their effect was strongly dependent on both concentration applied and culture period duration. The replacement of anticytokinins with benzyladenine in the medium resulted in overcoming of the bud break suppression. Both compounds significantly inhibited bud outgrowth as well.