3-DIMENSIONAL ANALYSIS OF THE SENESCENCE PROGRAM IN RICE (ORYZA-SATIVA L.) COLEOPTILES - INVESTIGATIONS BY FLUORESCENCE MICROSCOPY AND ELECTRON-MICROSCOPY

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescen ce and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The inter veinal region in cross-sections taken 1 mm from the tip of the coleopt ile was selected for this analysis. Fluorescence microscopy using samp les embedded in Technovit 7100 resin, electron microscopy and immunoel ectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the followi ng order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condens ation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase, dilation of the thyla koid membranes, increase in size and number of osmiophilic globules, c ondensation of the cytoplasm; (iii) disorganization of the nucleus, de generation of the tonoplast; (iv) complete loss of the cytoplasmic com ponents, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mito chondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. Thi s occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests th at cpDNA degradation involves rough digestion first, rather than rapid , direct decomposition of the DNA into nucleotides. The fragmented cpD NA is then dispersed throughout the chloroplast and cytoplasm.