Programmed cell death during pollination-induced petal senescence in petunia

Petal senescence, one type of programmed cell death (PCD) in plants, is a genetically controlled sequence of events comprising its final developmental stage. We characterized the pollination-induced petal senescence process in Petunia inflata using a number of cell performance markers, including fresh/dry weight, protein amount, RNA amount, RNase activity, and cellular membrane leakage. Membrane disruption and DNA fragmentation with preferential oligonucleosomal cleavage, events characteristic of PCD, were found to be present in the advanced stage of petal senescence, indicating that plant and animal cell death phenomena share one of the molecular events in the execution phase. As in apoptosis in animals, both single-stranded DNase and double-stranded DNase activities are induced during petal cell death and are enhanced by Ca(2+). In contrast, the release of cytochrome c from mitochondria, one commitment step in signaling of apoptosis in animal cells, was found to be dispensable in petal cell death. Some components of the signal transduction pathway for PCD in plants are likely to differ from those in animal cells.     

Temporal rhythm of petal programmed cell death in Ipomoea purpurea

• Flowers are the main sexual reproductive organs in plants. The shapes, colours and scents of corolla of plant flowers are involved in attracting insect pollinators and increasing reproductive success. The process of corolla senescence was investigated in Ipomoea purpurea (Convolvulaceae) in this study.
• In the research methods of plant anatomy, cytology, cell chemistry and molecular biology were used.
• The results showed that at the flowering stage cells already began to show distortion, chromatin condensation, mitochondrial membrane degradation and tonoplast dissolution and rupture. At this stage genomic DNA underwent massive but gradual random degradation. However, judging from the shape and structure, aging characteristics did not appear until the early flower senescence stage. The senescence process was slow, and it was completed at the late stage of flower senescence with a withering corolla.
• We may safely arrive at the conclusion that corolla senescence of I. purpurea was mediated by programmed cell death (PCD) that occurred at the flowering stage. The corolla senescence exhibited an obvious temporal rhythm, which demonstrated a high degree of coordination with pollination and fertilization.

     

Naphthoquinones as allelochemical triggers of programmed cell death

Juglone and plumbagin are plant bioactive derivatives of 1,4-naphthoquinone occurring in plants, whereas lots of these plants belong to invasive species. Clarifying of action of juglone and plumbagin applied on plant cell model represented by tobacco BY-2 cells was the basic aim of this work. It was shown that naphthoquinones are able to induce various structural, functional and enzymatic changes leading to processes of apoptic-like cell death. Using dihydroethidium as fluorescent probe the mechanism of naphthoquinones action was explained. They are able to generate reactive oxygen species, which play important role in processes of programmed cell death. Disruption of mitochondrial respiratory chain was detected too. This study shown that mechanism of naphthoquinones action to plant cells is very complex and predestine them to be very effective compounds in plant competition fight.     

1-Butanol triggers programmed cell death in Populus euphratica cell cultures

1-Butanol, which is a specific inhibitor of phospholipase D, usually inhibits phosphatidic acid (PA) production and blocks the PA-dependent signaling pathway under stress conditions. However, the effects of 1-butanol on plant cells under non-stress condition are still unclear. In this study, we report that 1-butanol induced a dose dependent cell death in poplar (Populus euphratica) cell cultures. In contrast, the control 2-butanol and ethanol had no effects on cell viability. 1-Butanol-treated cells displayed hallmark features of programmed cell death (PCD), such as shrinkage of the cytoplasm, DNA fragmentation, condensed or stretched chromatin and the activation of caspase-3-like protease. Exogenous application of PA markedly inhibited the 1-butanol-induced PCD. 1-Butanol also caused a burst of mitochondrial H₂O₂ ([H₂O₂]_mit) that was usually accompanied by a loss of mitochondrial membrane potential (?Ψm). Supplement of PA, antioxidant enzyme (catalase) and antioxidant (ascorbic acid) reversed this effect. Moreover, a significant increase of nitric oxide (NO) was observed in 1-butanol-treated poplar cells. This NO burst was suppressed by PA or inhibitors of NO synthesis. Further pharmacological experiments indicate that the burst of NO contributed to the 1-butanol-induced inhibition of antioxidant enzymes and subsequent H₂O₂-dependent PCD. In conclusion, we propose that 1-butanol is a potent inducer of PCD in plants and this process is regulated by the PA, NO and H₂O₂.     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

9-O-acetylated GD3 triggers programmed cell death in mature erythrocytes

An acetylated modification of a tumor-associated ganglioside GD3 (9-O-AcGD3) is expressed in certain tumors and present during early stages of development in different tissues. However, the status and the role of 9-O-AcGD3 in the erythroid progenitor cells remain unexplored. Here, we report the level of 9-O-AcGD3 during erythropoiesis in bone marrow is down regulated during maturation. Signaling via 9-O-AcGD3 induces alteration of morphology and membrane characteristics of mature erythrocytes. This process also induces, a cell death program in these erythrocytes even in the absence of nucleus, mitochondria and other cell organelles sharing features of apoptosis in nucleated cells like membrane alterations, vesicularization, phosphatidyl serine exposure, activation of cysteine proteases like caspase-3. This is the first report of a programmed cell death pathway in mature erythrocytes, triggered by 9-O-AcGD3 contrary to their anti-apoptotic role in lymphoblasts, which suggests a cell specific role of this O-acetyl ester of GD3.     

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.     

Protein changes during programmed cell death in tobacco

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.     

A ligand-receptor pair that triggers a non-apoptotic form of programmed cell death

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.     

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotiana tabacum) flowers

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.     

Monitoring the mitochondrial transmembrane potential with the JC-1 fluorochrome in programmed cell death during mesophyll leaf senescence

Summary. Analysis of the mitochondrial transmembrane potential (_m) with the help of the JC-1 fluorochrome (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide) during mesophyll leaf senescence was performed in order to determine whether a reduction of _m takes place during mesophyll senescence and whether plant mitochondria, like mammalian ones, might be involved in the induction of programmed cell death. Fluorescence analysis of mesophyll protoplasts of Pisum sativum in a confocal microscope, fluorescent spectra analysis and time dependence of fluorescence intensity of monomers and of J-aggregates revealed that JC-1 is incorporated and accumulated specifically in plant mitochondria. Analysis of _m during mesophyll protoplast senescence revealed that two subpopulations of mitochondria which differ in _m exist in all analyzed stages of leaf senescence. The first subpopulation contains mitochondria with red fluorescence of J-aggregates due to an unperturbed high _m. The second subpopulation comprises mitochondria with green fluorescence of monomers due to a low _m, proving total depolarization of mitochondrial membranes. Fluorescence analysis demonstrated that even in the latest analyzed stages of leaf senescence, mitochondria with a high _m still exist. Fluorometric measurements revealed that the fluorescence intensity of J-aggregates decreases with the age of plants, which indicates that a reduction of _m during the mesophyll senescence process takes place; however, it does not take place within the whole population of mitochondria of the same protoplast. The reason of this can be due to a dramatic reorganization of mitochondria in mesophyll cells and the appearance of large mitochondria with local heterogeneity of _m in the oldest analyzed stages. All mitochondria in every stage of senescence maintained their membrane organization even when their size, distribution, and spatial organization in protoplasts changed dramatically. We stated that the reduction of _m does not directly induce programmed cell death in mesophyll cells, as opposed to animal apoptosis.Correspondence and reprints: Department of Plant Anatomy and Cytology, Institute of Experimental Biology of Plants, Warsaw University, Miecznikowa 1, 02-096 Warszawa, Poland.     

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.     

Aspects of programmed cell death during early senescence of barley leaves: possible role of nitric oxide

Summary. Leaf senescence is a highly coordinated process which involves programmed cell death (PCD). Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are less well known. In this study, we correlated condensation of chromatin and nuclear DNA (nDNA) fragmentation, two main features of PCD during early senescence in barley leaves, with the appearance of nitric oxide (NO) within leaf tissue. With the help of the alkaline version of the comet assay, together with measurements of nDNA fluorescence intensity, we performed a detailed analysis of the degree of nDNA fragmentation. We localised NO in vivo and in situ within the leaf and photometrically measured its concentration with the NO-specific fluorochrome 4-amino-5-methylamino-2′,7′-difluorofluorescein. We found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order: first nDNA fragmentation, in leaves of 6-day-old seedlings, and later chromatin condensation, in the apical part of leaves from 10-day-old seedlings. PCD did not start simultaneously even in neighbouring cells and probably did not proceed at the same rate. NO was localised in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. Localisation of NO in vascular tissue and in a large number of mesophyll cells during the senescence process might imply its transport to other parts of the leaf and its involvement in signalling between cells. The fact that the highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence and lower concentrations were found during later stages might suggest that NO plays an inductive role in PCD. Correspondence: A. Mostowska, Department of Plant Anatomy and Cytology, Institute of Experimental Biology of Plants, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.     

Evolution of LDH isozymes during programmed cell death

• 1.1. Lactic dehydrogenase dehydrogenase isozymes and other respiratory enzymes were studied in degenerating intersegmental muscles of Manduca sexta and Antheraea polyphemus.
• 2.2. Total activities of the different enzymes (isocitric dehydrogenase, malic dehydrogenase, catalase, lactic dehydrogenase) decline at varying rates, starting before the rapid phase of involution.
• 3.3. One isozyme of LDH, an M-type isozyme, increases several-fold during the final three days prior to the emergence of the insect.
• 4.4. The same isozyme appears very transiently or not at all in muscles which do not break down, and is present in degenerating silk glands at the time of their most rapid involution.
• 5.5. The data suggest that limitation of oxidative metabolism plays a role in the involution of the muscles.

     

Detection of programmed cell death using fluorescence energy transfer.

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.     

Victorin triggers programmed cell death and the defense response via interaction with a cell surface mediator

The host-selective toxin victorin is produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Victorin has been shown to bind to the P protein of the glycine decarboxylase complex (GDC) in mitochondria, and induce defense-related responses such as phytoalexin synthesis, extracellular alkalization and programmed cell death. However, evidence demonstrating that the GDC plays a critical role in the onset of cell death is still lacking, and the role of defense-like responses in the pathogenicity has yet to be elucidated. Here, cytofluorimetric analyses, using the fluorescein (VicFluor) or bovine serum albumin-fluorescein derivative of victorin (VicBSA), demonstrated that victorin-induced cell death occurs before these conjugates traverse the plasma membrane. As with native victorin, VicBSA clearly elicits apoptosis-like cell death, production of phytoalexin, extracellular alkalization, and generation of nitric oxide and reactive oxygen intermediates. These results suggest that the initial recognition of victorin takes place on the cell surface, not in mitochondria, and leads to the activation of a battery of victorin-induced responses. Pharmacological studies showed that extracellular alkalization is the essential regulator for both victorin- and VicBSA-induced cellular responses. We propose a model where victorin may kill the host cell by activating an HR-like response, independent of the binding to the GDC, through ion fluxes across the plasma membrane.