RESUMEN
A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants.
Asunto(s)
Apoptosis/fisiología , Cromatina/química , Nicotiana/citología , Nicotiana/fisiología , Plantas Tóxicas , Ácidos/farmacología , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Fluorescencia , Colorantes Fluorescentes , Histonas/química , Histonas/efectos de los fármacos , Histonas/metabolismo , Hidrólisis/efectos de los fármacos , Cinética , Microscopía Confocal/métodos , Propidio , Sensibilidad y Especificidad , Nicotiana/químicaRESUMEN
An early indicator of apoptosis in mammalian cells is the loss of the phospholipid membrane asymmetry of the cell. This results in exposure of phosphatidylserine on the outer surface of the plasma membrane. This change in membrane asymmetry can be analysed using annexin V. A further feature of apoptosis, DNA breaks, can be measured by the TUNEL assay. Using flow cytometry, we have identified both of these features in HL-60 cells and by modifying the techniques for plants, we have verified that these features also occur in plant cells undergoing apoptosis. In both plant and HL-60 cells, apoptosis was induced by treatment with camptothecin (1 microM). Annexin V binding was found to be an early indicator of apoptosis, occurring prior to the detection of DNA strand breaks as monitored by the TUNEL assay. In plant cells, chromatin condensation was detected prior to the detection of annexin V. No loss in membrane integrity occurred with apoptotic cells in comparison with necrotic cells. Our findings indicate that a form of apoptosis occurs in plants, with flow cytometric characteristics similar to those of apoptosis in HL-60 cells.
Asunto(s)
Anexina A5/metabolismo , Apoptosis , Camptotecina/farmacología , ADN/efectos de los fármacos , Fragmentación del ADN , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Citometría de Flujo , Técnicas Genéticas , Células HL-60 , Humanos , Plantas Tóxicas , NicotianaRESUMEN
White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.