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 M). Key terms: annexin V; apoptosis; plants; TUNELThere is little published information available on the nature of the apoptotic pathway in plant cells. It is not known how plant cell death is controlled, but it is likely that apoptosis is involved in plant response to environmental stresses, in the hypersensitive reaction to pathogen attack (3,9), and in plant senescence (O'Brien, unpublished data). In addition, apoptosis is likely to be involved in pollen tube growth down the style of flowers, xylem (and hence wood) development, and other developmental processes where cell death occurs as part of normal tissue and organ specialization (10).In previous work on Pinus leaf cells (15), we found a decrease in relative nuclear DNA (nDNA) fluorescence, suggesting chromatin condensation, and an appearance of a subdiploid peak indicative of nDNA fragmentation, both features of apoptosis (2). We hypothesized that these changes were indicators of pathways of apoptosis in plants and, investigating further, compared apoptotic features of Nicotiana cells with those of HL-60 cells. An unresolved question regarding chromatin condensation in HL-60 cells is its timing with respect to nucleosomal fragmentation as measured by the TUNEL assay. We wished to compare this with the situation in plant cells in order to align early indications of chromatin condensation with other features of apoptosis. In this regard, it was particularly important to have indicators of apoptosis earlier than the detection of a subdiploid nuclear peak. The candidate assays for this were the TUNEL assay, to identify nDNA fragmentation prior to the detection of an apoptotic peak (8), and second, annexin V binding. Recently, cell surface exposure of phosphatidylserine (PS) has been identified as an early indicator of apoptosis in mammalian cells. It occurs in a wide range of cell types in response to a variety of apoptosis-inducing signals (5,12,13,19,20). Using a fluorescent conjugate of annexin V, which binds preferentially to negatively charged phospholipids such as PS (1,18), it has been shown that PS exposure precedes nuclear changes and DNA fragmentation (13).In order to apply these protocols to plant cells, we modified the techniques used for monitoring apoptosis in *Correspondence to: Iona O'Brien, Mt.
White clover mosaic virus strain 0 (WCIMV-0), 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 necesy for cell-to-cell movement in plants but not for replication. The WCIMV-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 Nicodiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WCIMV-O at 1 jug of WCIMV 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 WC1MV strains (0, M, and J) or with WCIMV-O RNA at 10 pg/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 pg of WCIMV virions per ml. In i , the three 13* tnsenic 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 rsistnt to tobacco mosaic virus of the Tobamovius 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.
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