Studies on Plastid-Nuclei (Nucleoids) in <italic>Nicotiana tabacum</italic> L. II. Disassembly and Reassembly of Proplastid-Nuclei Isolated from Cultured Cells

Abstract

The effects of various concentrations of NaCl on the compact structure of proplastid-nuclei (pp-nuclei) isolated from cultured tobacco cells were examined by fluorescence microscopy using a DNA-specific fluorochrome, 4, 6-diamidino-2-phenylindole. Simultaneously, behavior of the four proplastid DNA-binding proteins (mol wt: 69kDa, 31 kDa, 30kDa and 14kDa) identified previously (Nemoto et al. 1988) was investigated by SDS-polyacrylamide gel electrophoresis. When the concentration of NaCl was increased, the isolated pp-nuclei were gradually dispersed, and at concentrations greater than 0.4 M NaCl they were almost completely dispersed. During this disassembly process, the 31-kDa and 30-kDa proteins dissociated from the pelletable pp-nuclear fraction to the supernatant at 0.1 M NaCl, whereas the 69-kDa and 14-kDa proteins dissociated to the supernatant only at 0.4 M NaCl. When the concentration of NaCl was decreased again by dialysis, the pp-nuclei which had been dispersed by 2 M NaCl were gradually reassembled into compact structures which were almost identical to the original pp-nuclei. During this reassembly process, the 69-kDa and 14-kDa proteins first returned to the pellet fraction, and subsequently the 31-kDa and 30-kDa proteins moved into the pellet. This behavior of the proplastid DNA-binding proteins strongly suggests that the association of these proteins with plastid-DNA is responsible for the compact organization of pp-nuclei. In addition, it was indicated that the 69-kDa and 14-kDa proteins are more tightly bound to plastid-DNA than are the 31-kDa and 30-kDa proteins.