Studies on Plastid-Nuclei (Nucleoids) in <italic>Nicotiana tabacum</italic> L. III. Isolation of Chloroplast-Nuclei from Mesophyll Protoplasts and Identification of Chloroplast DNA-Binding Proteins

Abstract

We have developed a method of isolating morphologically intact chloroplast-nuclei (nucleoids) in large quantities from mesophyll protoplasts of Nicotiana tabacum L. cv. Bright Yellow-2. The isolated chloroplast-nuclei (cp-nuclei) were dispersed by the treatments with proteinase K, 2 M NaCl and 2 M KCL, which suggested that the cp-nuclei are compactly organized by an electrostatic interaction between the chloroplast-DNA and some protein(s). However, the four proplastid DNA-binding proteins identified previously (Nemoto et al. 1988) were not found in the cp-nuclei, and a different set of DNA-binding proteins (mol wt: 35 kDa, 28 kDa and 26 kDa) was detected in the cp-nuclei by a DNA-binding assay. On the other hand, the chloroplast-DNA was not different from the proplastid-DNA. These findings indicate that the cp-nuclei are constituted from the plastid-DNA and the chloroplast-specific DNA-binding proteins. This suggests that the DNA-binding proteins in proplastids are dynamically replaced with the chloroplast DNA-binding proteins during the differentiation of plastids from proplastids to chloroplasts. The change of DNA-binding proteins may be involved in the morphological change of plastid-nuclei and/or the regulation of plastid-DNA replication and gene expression during the differentiation process of plastids.