Modulation of Programmed Cell Death in a Model System of Xylogenic Zinnia (Zinnia Elegans) Cell Culture

Programmed cell death is an integral part of the latest stage of differentiation of the tracheary elements of plant xylem vascular system. In this study, by applying a pharmacological approach with specific peptide inhibitors, we have elucidated the involvement of plant caspase-like proteases in cell death signaling during vessel elements formation in xylogenic zinnia (Zinnia elegans) cell culture. Cell culture was isolated from zinnia leaves and exposed to chemical treatments. Broad spectrum caspase inhibitor, caspase 1 and caspase-3 inhibitors were administrated to the cell culture where the cells were induced to differentiate with the addition of 1 mg/L benzylaminopurine and 0.1 mg/L ¿-naphthalene-acetic acid. The differentiation was substantially suppressed in presence of 100 nM concentration of the inhibitors. This indicates that the re-differentiation in cultured mesophyll zinnia cells is a PCD event that most probably occurs through signaling involving caspase-like proteases. The reported results are the first that provide evidence for participation of plant caspase-related enzymes in xylogenesis in zinnia. Laser scanning confocal microscopy revealed differentiation associated structural cellular changes, including formation of large vacuole, secondary wall thickenings, tonoplast rupture and cell autolysis to form hollow dead tracheary elements.

Cadmium-induced programmed cell death signaling in tomato suspension cells

Here we present a summary of our study on cadmium-induced cell death signaling in a model system of suspension-cultured tomato cells. Exposure of the cells to CdSO4 induced typical for PCD (cytoplasm shrinkage and nuclear condensation) morphological changes of the dead cells. Ethylene and hydrogen peroxide were established to increase in response to Cd stress. By inhibitory studies we have demonstrated that CdSO4 induces cell death in tomato suspension cells that involve plant caspase-like proteases, indicating that Cd-triggered cell death is a form of programmed cell death. It is also shown that serine peptidases, oxidative stress, lipid signaling and ethylene are players in the Cd-induced cell death signaling. Cd-induced cell death in tomato suspension cells was categorised as non-lysosomal type

Alternaria alternata AT Toxin Induces Programmed Cell Death in Tobacco

Detached tobacco leaves were infiltrated with an AT toxin preparation from the foliar pathogen Alternaria alternata tobacco pathotype. The AT toxin preparation caused formation of necrotic lesions within 5 days post-infiltration in a concentration-dependent manner. Cell death was accompanied by increased levels of the stress metabolites hydrogen peroxide, malondialdehyde, free proline and by enhanced total protease activity. Lesion development and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with caspase-specific peptide inhibitors (irreversible caspase-1 inhibitor acyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and the broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB)), the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethylketone and the polyamine spermine. Extensive accumulation of reactive oxygen species (ROS), as determined by staining with 3-3'-diaminobenzidine and 2',7'-dichlorofluorescein diacetate, was found in the AT toxin-challenged lesions. The data show that AT toxin-induced cell death in tobacco is a type of programmed cell death in which caspase-like proteases and ROS signalling play a prominent role

A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin-induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed

Chemical -induced apoptotic cell death in tomato cells : involvement of caspase-like proteases

A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants

Xylanse-Induced cell death events in detached tobacco leaves

Plant-pathogen interactions are associated with plant defense mechanism known as hypersensitive response (HR), which is a form of programmed cell death (PCD). In the present work we have tested the potency of chemicals, proven as PCD inhibitors in other systems, to prevent the spread of cell death in detached tobacco leaves challenged with ß- xylanase from Aspergillus awamori K1. Xylanase induced cell death that was accompanied by electrolyte leakage and increased levels of the stress metabolites hydrogen peroxide and malondialdehyde. Lesion development, ion leakage and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB), cysteine protease inhibitor iodoacetamide (IA), serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), the antioxidant l-galactone-¿-lactone (L-gal) and the ethylene blocker (S)-trans-2-Amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride (AVG). Massive ROS accumulation, as determined by staining with 3-3¿-diaminobenzidine and 2¿,7¿-dichlorofluorescein diacetate, occurred in xylanase-infiltrated lesions and was substantially reduced by the inhibitors. To the best of our knowledge, this is the first report showing that ß- xylanase produced by Aspergillus awamori K1 induces cell death response in tobacco and proteolysis, ROS and ethylene are involved in the mediation of the signaling

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

Item does not contain fulltextCadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 23 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub mu M concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H2O2) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H2O2 production and activation of PLC, PLD and ethylene signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved

Mastoparan-Induced Cell Death Signalling in Chlamydomonas Reinhardtii

The present study was focused on the elucidation of stress-induced cell death signaling events in the unicellular alga Chlamydomonas reinhardtii exposed to treatment with wasp venom mastoparan. By applying pharmacological approach with specific inhibitors, we have investigated the involvement of ethylene, nitric oxide and lipid signaling in MP-treated C. reinhardtii. Superior sensitive laser-based detectors were used for real-time measurement of trace amounts of ethylene and nitric oxide. The morphological features in the cells undergoing programmed cell death were detected by using laser-scanning confocal microscopy. Data showed that mastoparan induces programmed cell death in C. reinhardtii 137 C(+) that is associated with phospholipid signaling, including phospholipases C and D, ethylene and nitric oxide and, the dead cells express apoptotic-like cellular disintegration, involving cytoplasm shrinkage and condensation of the nucleus