Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants.
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.
O'Brien
I E
IE
Horticulture and Food Research Institute of New Zealand, Auckland. iobrien@hort.cri.nz
Reutelingsperger
C P
CP
Holdaway
K M
KM
D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000187:drug effects; D053938:DNA Fragmentation; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D005821:Genetic Techniques; D018922:HL-60 Cells; D006801:Humans; D010947:Plants, Toxic; D014026:Nicotiana
D017304:Annexin A5; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D004247:DNA; D002166:Camptothecin