RESUMEN
BACKGROUND: Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the obscuring effect of population heterogeneity that occurs with the use of other techniques. METHODS: Cell viability, plasma membrane integrity, oxidative activity, intracellular calcium and pH, cell wall thickening, the possible role of microtubule rearrangement, chromatin condensation, and DNA breakdown were followed by flow cytometry from the first stages of TE induction. RESULTS: TE differentiation could be enhanced and made more synchronous by a centrifugation step at 72 h after cell isolation. Size and shape changes were the first changes identified in differentiating cells, and these properties could be used to isolate differentiating populations by back-gating. Chromatin condensation and nDNA breakdown followed patterns characteristic of programmed cell death. CONCLUSIONS: We have used flow cytometry to characterize the morphological and physiological changes that occur during TE differentiation, and our findings indicate that this process is a form of autophagic PCD in which microtubule rearrangement appears to play a role.
Asunto(s)
Asteraceae/citología , Citometría de Flujo/métodos , Hojas de la Planta/citología , Asteraceae/fisiología , Autofagia , Calcio/análisis , Diferenciación Celular , Membrana Celular/fisiología , Forma de la Célula , Tamaño de la Célula , Supervivencia Celular , Células Cultivadas , Cromatina/fisiología , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Microtúbulos/fisiología , Estrés Oxidativo , Hojas de la Planta/química , Hojas de la Planta/fisiologíaRESUMEN
BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.
Asunto(s)
Apoptosis/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Nicotiana/fisiología , Protoplastos/efectos de los fármacos , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacología , Animales , Fragmentación del ADN , Proteínas HSP70 de Choque Térmico/genética , Calor , Potenciales de la Membrana/fisiología , Mitocondrias/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Nicotiana/citologíaRESUMEN
Programmed cell death (PCD) is an active way for plant cells marching to death, which plays an important role in plant development and stress responses. Cytological, biochemical, molecular and physiological methods for measuring plant PCD were reviewed. Application of flow cytometer to plant PCD research was also covered.
Asunto(s)
Apoptosis , Citometría de Flujo/métodos , Etiquetado Corte-Fin in Situ/métodos , Células Vegetales , Coloración y Etiquetado/métodos , Caspasas/metabolismo , Citocromos c/metabolismo , Fragmentación del ADN , ADN de Plantas/análisis , ADN de Plantas/genética , Plantas/genética , Plantas/metabolismoRESUMEN
BACKGROUND: We present evidence that in plant cells DNA damage induced by the topoisomerase trapping drug camptothecin induces oxidative stress via the mitochondrial respiratory chain. METHODS: Flow cytometry was used to analyse mitochondrial respiratory chain activity by simultaneous measurement of mitochondrial generation of reactive oxygen intermediates (ROI) and mitochondrial membrane potential (DeltaPsim), in live functioning sugarbeet protoplasts. The mitochondrial respiratory chain function was assessed by the addition of substrate and inhibitors to the digitonin-permeabilised protoplasts. Mitochondrial activity was also measured in protoplasts at different stages of induced apoptosis by camptothecin. RESULTS: The addition of camptothecin to sugarbeet protoplasts resulted in increased mitochondrial generation of ROI, occurring concurrently with the hyperpolarisation of the mitochondrial inner membrane. Subsequently, there was a decrease in mitochondrial-generated ROI, in association with a decrease in DeltaPsim. These camptothecin-treated digitonin-permeabilised protoplasts could be re-energised and ROI decreased by the addition of malic acid in the early stages of apoptosis induction, but not in the latter stages. There was a depolarisation of the mitochondrial inner membrane in the final stages of apoptosis. CONCLUSIONS: These results are consistent with impaired adenine nucleotide exchange across the mitochondrial membrane, suggesting that similar mechanisms regulate plant apoptosis as those described for mammalian apoptosis.
Asunto(s)
Apoptosis , Transporte de Electrón/fisiología , Mitocondrias/patología , Estrés Oxidativo , Células Vegetales , Camptotecina/farmacología , Daño del ADN , Digitonina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Indicadores y Reactivos/farmacología , Potenciales de la Membrana , Microscopía Confocal , Consumo de Oxígeno , Especies Reactivas de Oxígeno , Factores de TiempoRESUMEN
The clinical antitumor efficacy of nitrogen mustards such as chlorambucil may relate to their ability to cause programmed cell death (apoptosis), probably through their DNA cross-linking properties. In contrast, bisbenzimidazoles such as Hoechst 33342 interact noncovalently with the minor groove of DNA, and appear to cause apoptosis in a fundamentally different way, which may involve the inhibition of topoisomerase (topo) I enzymes. A series of DNA minor groove binding nitrogen mustards with selective DNA affinity and in vivo antitumor activity in animal models was studied. Although two examples of such compounds proved to inhibit topo I enzymes in vitro, they were equally toxic towards topo I-proficient and- deficient strains of yeast, suggesting that topo I inhibition was not involved in cell killing. Flow cytometric analysis of Chinese hamster cells highlighted the differences in the propensity to cause apoptosis by chlorambucil compared with Hoechst 33342, revealing two distinct apoptotic populations in cells treated with the latter drug. Unexpectedly, the bisbenzimidazole mustards showed a novel peak of apoptotic activity, distinct from that shown by either parent drug. Exploring these different mechanisms of apoptosis may provide new directions for the development of antitumor drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , ADN/metabolismo , Compuestos de Mostaza Nitrogenada/farmacología , Animales , Apoptosis/fisiología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células CHO , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Cricetinae , ADN/genética , HumanosRESUMEN
Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.